Botulinum toxin screening assays

ABSTRACT

Methods for detecting BoNT/A activity in a sample, methods for screening molecules able to compete with BoNT/A receptor binding, methods for reducing BoNT/A activity in a human and methods of marketing a neurotoxin capable of selectively binding to FGFR3 to a governmental or regional regulatory authority.

This application Ser. No. 12/545,647 application is a divisionalapplication of US non-provisional application No. 10/598,073 filed Aug.17, 2006, now U.S. Pat. 7,598,027, which is a 371 of PCT/US05/06421filed Feb. 23, 2005, which claims benefit of the US provisionalapplication 60/547,591 filed Feb. 24, 2004.

All of the publications cited in this application are herebyincorporated by reference herein in their entirety.

The myorelaxant properties of Botulinum toxins (BoNTs) are beingexploited in a wide variety of therapeutic and cosmetic applications,see e.g., William J. Lipham, COSMETIC AND CLINICAL APPLICATIONS OFBOTULINUM TOXIN (Slack, Inc., 2004). For example, CoNTs therapies areproposed for treating dystonia, see e.g., Kei Roger Aoki, et al., Methodfor treating Dystonia with Botulinum Toxin C to G, U.S. Pat. No.6,319,505 (Nov. 20, 2001); pain, see e.g., Kei Roger Aoki, et al.,Method for Treating Pain by Peripheral Administration of a Neurotoxin,U.S. Pat. No. 6,464,986 (Oct. 15, 2002); muscle injuries, see e.g.,Gregory F. Brooks, Methods for Treating Muscle Injuries, U.S. Pat. No.6,423,319 (Jul. 23, 2002); cardiovascular diseases, see e.g., Gregory F.Brooks, Methods for Treating Cardiovascular Diseases with BotulinumToxins, U.S. Patent Publication No. 2003/0185860 (Oct. 2, 2003);neuropsychiatric disorders, see e.g., Steven Donovan, TherapeuticTreatments for Neuropsychiatric Disorders, U.S. Patent Publication No.2003/0211121 (Nov. 13, 2003); lower back pain, see e.g., Kei Roger Aoki,et al., Botulinum Toxin Therapy for Lower Back Pain, U.S. PatentPublication No. 2004/0037852 (Feb. 26, 2004); as well as otherneuromuscular disorders, see e.g., Kei Roger Aoki, et al., MultipleBotulinum Toxins for Treating Neuromuscular Disorders and Conditions,U.S. Patent Publication No. 2001/0021695 (Sep. 13, 2001); Kei RogerAoki, et al., Treatment of Neuromuscular Disorders and Conditions withDifferent Botulinum, U.S. Patent Publication No. 2002/0010138 (Jan. 24,2002); Kei Roger Aoki, et al., Use of Botulinum Toxins for TreatingVarious Disorders and Conditions and Associated Pain, U.S. PatentPublication No. 2004/0013692 (Jan. 22, 2004). Additional proposed usesof BoNTs as biopharmaceutical neuromodulators has expanded to cover awide variety of treatments targeting certain disorders that lack aneuromuscular basis. For example, the effects on the autonomic nervoussystem has allowed the development of a Botulinum toxin serotype A(BoNT/A) therapy for treating axillary hyperhydrosis or sweating, andreports indicate BoNT/A may be an effective treatment for myofascialpain and tension, stroke, traumatic brain injury, cerebral palsy,gastrointestinal motility disorders, urinary incontinence cancer andmigraine headaches. Lastly, cosmetic and other therapeutic applicationsare widely known. In fact, the expected use of BoNTs in both therapeuticand cosmetic treatments of humans is anticipated to expand to an everwidening range of diseases and aliments that can benefit from themyorelaxant properties of these toxins.

The growing clinical and therapeutic use of botulinum toxinsnecessitates the pharmaceutical industry to use accurate assays for BoNTactivity in order to, for example, ensure accurate pharmaceuticalformulations and monitor established quality control standards. Inaddition, given the potential danger associated with small quantities ofBoNT in foodstuffs, the food industry requires BoNT activity assays, forexample, to validate new food packaging methods and to ensure foodsafety. Additionally, BoNT activity assays are useful in identifyingmodulators of BoNT activity, for example, modulators that reduce BoNTactivity which can be useful as a toxin antidote and modulators thatincrease BoNT activity which can be useful in creating more potent orlonger lasting pharmaceutical formulations. The present inventionprovides novel BoNT assays for detecting the presence or activity of aBoNT useful for various industries, such as, e.g. the pharmaceutical andfood industries, and provides related advantages as well.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic of the current paradigm of the BoNT/Aintoxication mechanism. This intoxication process can be described ascomprising four steps: 1) receptor binding, where BoNT/A binds to aBoNT/A receptor system initiates the intoxication process; 2) complexinternalization, where after BoNT/A binding, a vesicle containing atoxin/receptor system complex is endocytosised into the cell; 3) lightchain translocation, where multiple events are thought to occur,including changes in the internal pH of the vesicle, formation of achannel pore comprising the H_(N) domain of BoNT/A heavy chain,separation of the BoNT/A light chain from the heavy chain, enzymaticactivation of the light chain; and release of the activated light chainand 4) enzymatic target modification, where the activated light chain ofBoNT/A proteolytically cleaves its target SNARE substrates, such as,e.g., SNAP-25.

FIG. 2 shows a schematic of an FGFR3 and the alternatively spliced exonsthat result in FGFR3IIIb and FGFR3IIIc. The top diagram shows ageneralized drawing of a FGFR3. The extracellular domain comprises asignal peptide (box labeled SP), three Ig-like domains (loops labeledIgI, IgII and IgIII) and an acid box (box labeled acid). A singlemembrane spanning region comprises the transmembrane domain (box labeledTM). The cytoplasmic portion of the receptor comprises the tyrosinekinase domain. The middle diagram shows a generalized drawing of theexons encoding a FGFR3IIIb isoform, where exon 9 is spliced out from theprimary transcript during processing. The lower diagram shows ageneralized drawing of the exons encoding a FGFR3IIIc isoform, whereexon 8 is spliced out from the primary transcript during processing.

FIG. 3 shows the results of electroporation of PURE-A into HIT-T15cells. FIG. 3 a shows the results of an inhibition of insulin releaseassay. The graph indicates that the addition of glucose to 25 mM inducedinsulin secretion from untreated cells (control) and cells subjected toelectroporation without the addition of PURE-A (Electroporation NoPURE-A). However, HIT-T15 cells into which PURE-A was introduced(Electroporation PURE-A) showed a decrease in insulin secretion fromindicating these cells were unresponsive to induction of insulinsecretion. FIG. 3 b shows the results of a SNAP-25 cleavage assay.Western blot analysis identified the presence of a BoNT/A SNAP-25₁₉₇cleavage product in PURE-A treated cells (Electroporation PURE-A), butnot in either control (Control and Electroporation No PURE-A), withequal amounts of protein loaded per lane and probed with an antibodythat detects the BoNT/A SNAP-25₁₉₇ cleavage product.

FIG. 4 shows the affects of electroporation of HIT-T15 cells over time.FIG. 4 a shows the results on an inhibition release for insulin assaydemonstrating that the presence of the toxin delayed growth in HIT-T15cells when compared to controls, but toxin-treated cells were able toreplicate normally after a recovery period. FIG. 4 b shows a westernblot analysis demonstrating that cleavage of SNAP-25 was detected at alltime points tested when PURE-A was introduced into the cells, with equalamounts of protein loaded per lane and probed with an antibody thatdetects the BoNT/A SNAP-25₁₉₇ cleavage product.

FIG. 5 shows HIT-T15 cells, transformed with a human brain cDNA libraryand selected using magnetic beads to which BONT/A had been bound.Individual colonies are visible in the dish and are surrounded bymagnetic beads.

FIG. 6 shows the results of an assay of insulin release from HIT-T15cells containing the putative BONT/A receptor. Cells were exposed to 1nM PURE-A and assayed for inhibition of insulin release upon glucosestimulation.

FIG. 7 shows the analysis of two isolated HIT-T15 cell isolates C6 andC7. FIG. 7 a shows the reduction of insulin release in representativeHIT-T15 transformants C6 and C7 upon incubation with BONT/A. FIG. 7 bshows a western blot analysis demonstrating that cleavage of SNAP-25 wasdetected in clones C6 and C7 incubated with BONT/A, with equal amountsof protein loaded per lane and probed with an antibody that detects theBoNT/A SNAP-25₁₉₇ cleavage product.

FIG. 8 shows Western blot analysis identifying cells with high affinityuptake for a Clostridial toxin. FIG. 8 a shows a Western blot analysisused to identify cells capable of BoNT/A uptake. The blot shows fivecell lines treated with 1 nM of PURE-A overnight, with equal amounts ofprotein loaded per lane and probed with an antibody that detects theBoNT/A SNAP-25197 cleavage product. FIG. 8 b shows Western blot analysisused to evaluate the time necessary for BoNT/A uptake. The blots showeither Neuro-2A cells or SH-SY5Y cells treated with 1 nM of PURE-A forvarious lengths of time, with equal amounts of protein loaded per laneand probed with an antibody that detects the BoNT/A SNAP-25₁₉₇ cleavageproduct. FIG. 8 c shows a Western blot analysis used to evaluate theconcentration range necessary of BoNT/A uptake. The blots show Neuro-2Acells treated with a range of PURE-A concentrations overnight, withequal amounts of protein loaded per lane and probed with an antibodythat detects the BoNT/A SNAP-25₁₉₇ cleavage product.

FIG. 9 shows Western blot analysis evaluating the effects of gangliosidetreatments used to increase uptake of a botulinum toxin. FIG. 9 a showsa Western blot analysis evaluating the effects of ganglioside treatmenton the uptake of BoNT/A. The blot shows Neuro-2A cells treated withoutor with 25 μg/mL of GT1b (− or +) and exposed overnight to threedifferent concentrations of BoNT/A (12.5 pM, 25 pM or 50 pM), with equalamounts of protein loaded per lane and probed with an antibody thatdetects the BoNT/A SNAP-25₁₉₇ cleavage product. FIG. 9 b shows a Westernblot analysis evaluating the effects of ganglioside treatment on theuptake of BoNT/E. The blot shows Neuro-2A cells treated with either 25μg/mL of GT1b, GQ1b, GD1a, GD1b or GD3 and exposed for approximately 5hours to 14 nM of BoNT/E di-chain, with equal amounts of protein loadedper lane and probed with an antibody (SMI-81; Sternberger Monoclonals,Lutherville, Md.) that detects the uncleaved SNAP-25₂₀₆ substrate andthe BoNT/E SNAP-25₁₈₀ cleavage product.

FIG. 10 shows the results of a crosslinking experiment in Neuro-2A cellsusing a BoNT/A-SBED toxin. FIG. 10 a shows the isolation of a complex ofapproximately 250 kDa from Neuro-2A cells containing the 150 kDaneurotoxin cross-linked to the putative BONT/A receptor. Bands werevisualized with silver staining. FIG. 10 b shows a Western blot analysisused to identify a BoNT/A receptor. The blots shows the presence of asingle band corresponding to the 97 kDa FGFR3 (first panel) and twobands corresponding to the 150 kDa BoNT/A holotoxin and the 100 kDaBoNT/A heavy chain (second panel), with equal amounts of protein loadedper lane and probed with an antibody that detects either FGFR3 orBoNT/A.

FIG. 11 shows a Western blot analysis used to determine the presence ofFGFRs in five different cell lines. Only antibodies selectively bindingto FGFR3 detected bands that correlated with cell lines that contained aBoNT/A receptor.

FIG. 12 shows the results of a receptor competition experiment inNeuro-2a cells using PURE-A and FGF ligands. A western blot analysisshows that both FGF1 and FGF2 effectively competed with BoNT/A forbinding to the BoNT/A receptor, with equal amounts of protein loaded perlane and probed with antibody (SMI-81; Sternberger Monoclonals,Lutherville, Md.) that detects the uncleaved SNAP-25₂₀₆ substrate andthe BoNT/E SNAP-25₁₈₀ cleavage product. The appearance of the uncleavedSNAP-25₂₀₆ substrate was detected when as little as 1 nM of FGF ligandwas present and clearly visible when 5 nM of FGF ligands were present.Detectable levels of the BoNT/A SNAP-25₁₉₇ cleavage product was absentin FGF ligand treatments of 200 mM.

FIG. 13 shows the results FGFR3 phosphorylation studies in Neuro-2Acells. FIG. 13 a shows a Western blot analysis indicating the presenceof phosphorylated FGFR3 after exposure to FGF2 or BoNT/A. The blot showsNeuro-2A cells treated with either 5 nM FGF2 or 5 nM PURE-A for variouslengths of time, with equal amounts of protein loaded per lane andprobed with an antibody that detects FGFR3. FIG. 13 b shows a Westernblot analysis indicating the reduction of phosphorylated FGFR3 whenexposed to increasing amounts of DMBI. The blot shows Neuro-2A cellstreated with 5 nM FGF2 for 10 minutes, with equal amounts of proteinloaded per lane and probed with an antibody that detects phosphorylatedFGFR3. FIG. 13 c shows a Western blot analysis indicating the reductionof SNAP-25₁₉₇ cleavage product when exposed to increasing amounts ofDMBI. The blots show either Neuro-2A cells treated with 5 nM of PURE-Afor 10 minutes, with equal amounts of protein loaded per lane and probedwith an antibody that detects the BoNT/A SNAP-25₁₉₇ cleavage product.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the identification of a cell surfacereceptor to which BoNT/A selectively binds as the first step to theselective intoxication of a neuron. The present specification, in part,discloses that the Fibroblast Growth Factor Receptor 3 (FGFR3) is usefulas a BoNT receptor, such as, e.g., a BoNT/A receptor. In addition, thepresent disclosure identifies specific gangliosides which facilitatebinding of a BoNT to a BoNT receptor and the internalization of thesetoxins within a neural cell., such as, e.g., an increased binding ofBoNT/A for a BoNT/A receptor using a ganglioside like GT1b; and anincreased binding of BoNT/E for a BoNT/E receptor using a gangliosidelike GQ1b, GD1a, GD1b or GD3.

The present invention provides novel assays for detecting the presenceor absence of an active BoNT/A. The novel methods disclosed in thepresent specification reduce the need for animal-based toxicity studies,yet serve to analyze multiple toxin functions, namely, binding andcellular uptake of toxin, translocation into the cell cytosol, andprotease activity. As discussed further below, the novel methods of thepresent disclosure can be used to analyze crude and bulk samples as wellas highly purified dichain toxins and formulated toxin products andfurther are amenable to automated high throughput assay formats.

Aspects of the present invention provide methods of detecting BoNT/Aactivity by contacting a sample to a cell that contains an exogenousFGFR3 wherein said contacted cell is capable of BoNT/A intoxication anddetecting the presence of BoNT/A activity of said contacted cellrelative to a control cell, where a difference in said BoNT/A activityof said contacted cell as compared to said control cell is indicative ofBoNT/A activity. Other aspects of the present invention provide methodsof detecting BoNT/A activity by contacting a sample to a cell thattransiently contains an exogenous FGFR3 wherein said contacted cell iscapable of BoNT/A intoxication and detecting the presence of BoNT/Aactivity of said contacted cell relative to a control cell, where adifference in said BoNT/A activity of said contacted cell as compared tosaid control cell is indicative of BoNT/A activity. Other aspects of thepresent invention provide methods of detecting BoNT/A activity bycontacting a sample to a cell that stably contains an exogenous FGFR3wherein said contacted cell is capable of BoNT/A intoxication anddetecting the presence of BoNT/A activity of said contacted cellrelative to a control cell, where a difference in said BoNT/A activityof said contacted cell as compared to said control cell is indicative ofBoNT/A activity.

Other aspect of the present invention provide methods of reducing BoNT/Aactivity in a human comprising administering to said human apharmaceutical composition comprising a molecule that selectively bindsa FGFR3 wherein said selective binding reduces the ability of BoNT/A tobind to said FGFR3.

Other aspect of the present invention provide methods of screening for amolecule able to compete with BoNT/A for selective binding to cellssusceptible to BoNT/A intoxication by contacting said sample with acomposition comprising an FGFR3 and detecting whether said moleculeselectively binds said FGFR3, wherein selective binding of said moleculeto said FGFR3 indicates that said molecule is able to compete withBoNT/A for selective binding to cells susceptible to BoNT/Aintoxication, and wherein if said molecule is BoNT/A, said method doesnot comprise an LD₅₀ assay.

Other aspect of the present invention provide methods of marketing aneurotoxin capable of selectively binding to the same FGFR3 as BoNT/Acomprising obtaining marketing approval from a governmental or regionalregulatory authority for a therapeutic neurotoxin, wherein saidneurotoxin is assayed for selective binding to a cell comprisingcontacting said neurotoxin with a composition comprising a FGFR3 anddetecting whether said neurotoxin selectively binds said FGFR3, whereinselective binding of said neurotoxin to said FGFR3 indicates that saidneurotoxin is able to selective binding to cells susceptible to BoNT/Aintoxication and wherein if said molecule is BoNT/A, said method doesnot comprise an LD₅₀ assay; packaging said neurotoxin for sale in amanner consistent with the requirements of said regulatory authority,and selling said neurotoxin.

Other aspect of the present invention provide methods of marketing aneurotoxin capable of selectively binding to the same FGFR3 as BoNT/Acomprising obtaining marketing approval from a governmental or regionalregulatory authority for a therapeutic neurotoxin, wherein saidneurotoxin is assayed for selective binding to a cell comprisingcontacting said neurotoxin to a cell that contains an exogenous FGFR3wherein said contacted cell is capable of BoNT/A intoxication anddetecting the presence of BoNT/A activity of said contacted cellrelative to a control cell, where a difference in said BoNT/A activityof said contacted cell as compared to said control cell is indicative ofBoNT/A activity; packaging said neurotoxin for sale in a mannerconsistent with the requirements of said regulatory authority, andselling said neurotoxin.

BoNTs are each translated as a single chain polypeptide of approximately150 kDa that is subsequently cleaved by proteolytic scission within adisulphide loop by bacterial or tissue proteases. This posttranslationalprocessing yields a di-chain molecule comprising an approximately 50 kDalight chain (LC) and an approximately 100 kDa heavy chain (HC) heldtogether by a single disulphide bond and noncovalent interactions. Eachmature di-chain molecule comprises three functionally distinctdomains: 1) an enzymatic domain located in the LC that includes ametalloprotease region containing a zinc-dependent endopeptidaseactivity which specifically targets core components of theneurotransmitter release apparatus; 2) a translocation domain containedwithin the amino-terminal half of the HC (H_(N)) that facilitatesrelease of the toxin from intracellular vesicles into the cytoplasm ofthe target cell; and 3) a binding domain found within thecarboxy-terminal half of the HC (H_(C)) that determines the bindingactivity and binding specificity of the toxin to the receptor complexlocated at the surface of the target cell.

The binding, translocation and enzymatic activity of these threefunctional domains are all necessary for toxicity. While all details ofthis process are not yet precisely known, the overall cellularintoxication mechanism whereby BoNTs enter a neuron and inhibitneurotransmitter release is similar, regardless of type. Although theapplicants have no wish to be limited by the following description, theintoxication mechanism can be described as comprising four steps: 1)receptor binding, 2) complex internalization, 3) light chaintranslocation, and 4) enzymatic target modification (see FIG. 1). Theprocess is initiated when the H_(C) domain of a BoNT binds toBoNT-specific receptor complex located on the plasma membrane surface ofa target cell. The binding specificity of a receptor complex is thoughtto be achieved, in part, by specific combinations of gangliosides andprotein receptors that appear to distinctly comprise each BoNT/Areceptor complex. Once bound, the BoNT/receptor complexes areinternalized by endocytosis and the internalized vesicles are sorted tospecific intracellular routes. The translocation step appears to betriggered by the acidification of the vesicle compartment. This processseems to initiate two important pH-dependent structural rearrangementsthat increase hydrophobicity and promote enzymatic activation of thetoxin. Once activated, light chain endopeptidase of the toxin isreleased from the intracellular vesicle into the cytosol where itspecifically targets one of three known core components of theneurotransmitter release apparatus. There of these core proteins,vesicle-associated membrane protein (VAMP)/synaptobrevin,synaptosomal-associated protein of 25 kDa (SNAP-25) and Syntaxin, arenecessary for synaptic vesicle docking and fusion at the nerve terminaland constitute members of the soluble N-ethylmaleimide-sensitivefactor-attachment protein-receptor (SNARE) family. The selectiveproteolysis of synaptic SNAREs accounts for the total block ofneurotransmitter release caused by clostridial toxins in vivo. The SNAREprotein targets of clostridial toxins are common to exocytosis in avariety of non-neuronal types; in these cells, as in neurons, lightchain peptidase activity inhibits exocytosis, see, e.g., Yann Humeau etal., How Botulinum and Tetanus Neurotoxins Block NeurotransmitterRelease, 82(5) Biochimie. 427-446 (2000); Kathryn Turton et al.,Botulinum and Tetanus Neurotoxins: Structure, Function and TherapeuticUtility, 27(11) Trends Biochem. Sci. 552-558. (2002); M. Zouhair Atassi,Basic and Therapeutic Aspects of Botulinum and Tetanus Toxins, (Dirk W.Dressler & Joseph J. Jankovic eds., 2003); Giovanna Lalli et al., TheJourney of Tetanus and Botulinum Neurotoxins in Neurons, 11(9) TrendsMicrobiol. 431-437, (2003).

The three-dimensional crystal structures of BoNT/A indicate that thethree functional domains of the toxin are structurally distinct, seee.g., Humeau et al., supra, (2000), Turton et al, supra, (2002); andLalli et al., supra, (2003). The HEXXH consensus motif of the lightchain forms the tetrahedral zinc binding pocket of the catalytic sitelocated in a deep cleft on the protein surface that is accessible by achannel. This conserved zinc binding motif binds at least one zinc atomnecessary for its catalytic function. The structure of the H_(N) andH_(C) domains consists primarily of β-sheet topologies that are linkedby a single α-helix. The H_(N) domain comprises a β-barrel, jelly-rollfold that resembles the carbohydrate binding moiety found in lectinssuggesting that this domain may recognize oligosaccharide-containingmolecules and play a role in the intracellular sorting. In addition toits overall structural similarity with lectins, the H_(N) domain alsocontains two distinct structural features suggesting functions. First,the H_(N) domain contains a pair of long amphipathic helices thatresemble the coiled-coil motif found in some viral proteins. In viruses,these helices assist in fusing the viral membrane to the cellularmembrane of the host, suggesting that the coiled-coil region may assistin inserting the H_(N) domain into the membrane of an intracellularvesicle. Second, a long loop called the ‘translocation belt,’ wrapsaround a large negatively charged cleft of the light chain that blocksaccess of the zinc atom to the catalytic-binding pocket of active site.The H_(C) domain contains a ganglioside-binding site and a five residueganglioside-binding motif. These regions adopt a modified β-trefoil foldstructure which forms four distinct carbohydrate binding regionsbelieved to mediate the binding to specific carbohydrate containingacceptor molecules on the cell surface. Consistent with this function,the H_(C) domain exhibits the highest sequence divergence betweenclostridial toxins which may account for the distinct binding propertiesand sorting schemes of TeNT and BoNTs. The H_(C) domain tilts away fromthe H_(N) domain exposing the surface loops and making them accessiblefor binding. No contact seems to occur between the light chain and theH_(C) domain. The N-terminus of the H_(C) region presents a jelly-rollarchitecture related to that of the S-lectins, a carbohydrate-bindingfamily of proteins. By contrast, the C-terminus of H_(C) is in a pseudothreefold trefoil conformation that presents structural similarity tothe sequentially unrelated interleukins-1α and 1β, Kunitz-type trypsininhibitors, as well as fibroblast growth factors (FGF). These proteins,mostly β-proteins, are involved in protein-protein interactions.

Cell surface gangliosides appear to be part of the receptor system forBoNT/A and appear to participate in binding of the toxin to its BoNT/Areceptor. Although toxin binding is not strictly dependent on thepresence of gangliosides, the presence of specific gangliosides appearsto be required for high affinity binding. In particular, BoNTs have beenobserved to interact in vitro and in vivo with polysialogangliosides,especially those of the G1b series (GD1a, GD1b, GD3, GQ1b, or GT1b),see, e.g., Jane L. Halpern & Elaine A. Neale, Neurospecific binding,internalization, and retrograde axonal transport, 195 Curr. Top.Microbiol. Immunol. 221-241 (1995). Preincubation of the toxin withthese gangliosides protects the neuromuscular junction (NMJ) of micefrom BoNT toxicity. High-affinity, trypsin-sensitive, BoNT-binding siteswere found in isolated synaptosomes, see, e.g., R. S. Williams et al,Radioiodination of botulinum neurotoxin type A with retention ofbiological activity and its binding to brain synaptosomes. 131(2) Eur.J. Biochem. 1437-1445 (1983). Since lectins with high affinity forsialic acid antagonize the binding of BoNTs, their protein receptors maybe glycoproteins. Receptors for BoNTs would direct them to acidicvesicles allowing the translocation of the LC into the cytosol of theneuron. The amino acid sequence at the C-terminus of H_(C) is poorlyconserved among different clostridial neurotoxins, and competitionexperiments have shown that different BoNT serotypes bind to differentprotein receptors on the surface of neuronal cells. This analysis istherefore consistent with the hypothesis that BoNTs neurotoxins bind toreceptor systems comprising at least two components; a protein componentand a carbohydrate component.

Based on these findings, and as the present disclosure provided herein,the Applicants have discovered that cells expressing the fibroblastgrowth factor receptor 3 (FGFR3) can bind BoNT/A. Internalization of thetoxin can be followed when these cell lines are exposed to the toxin.Moreover, BoNT/A internalization is inhibited in a dose-dependent mannerwhen FGF, such as, e.g., FGF1, FGF2, FGF4, FGF8 and FGF9, is added atincreasing concentrations. Cells tested by the Applicants that did notdisplay the FGFR3 receptor were unable to internalize the toxin,although when subjected to electroporation in the presence of BoNT/A,the intracellular cleavage of SNAP-25 could be detected, indicating thatthe endopeptidase activity of the toxin remained intact, and that thecells remained susceptible to the endopeptidase. In addition, theApplicants have found that pre-treatment with the polysialogangliosideGT1b increases BoNT/A cellular uptake.

Fibroblast growth factors (FGF) participate in many developmental,differentiation and growth and repair processes of cells through complexcombinatorial signaling pathways. Presently, at least 23 ligands(FGF1-23) are known to signal through a family of five transmembranetyrosine kinase FGF receptors (FGFR1-4). The amino acid sequenceidentity is highly conserved between FGFR family members and each sharea characteristic structural organization. The extracellular portion ofFGFRs comprise an amino-terminal hydrophobic signal peptide, threeIg-like domains (IgI, IgII and IgIII) and an acid box domain ofapproximately eight acidic residues, followed by a single hydrophobictransmembrane domain, which in turn is followed by an intracellulartyrosine kinase domain (see FIG. 2). Affinity of FGFRs for their ligandsis highly diverse with different affinities for each family member ofgrowth factors, see, e.g., C. J. Powers et al., Fibroblast growthfactors, their receptors and signaling 7(3) Endocr. Relat. Cancer.165-197 (2000). Table 1 lists some of the known FGF-FGFR signalingrelationships of various FGFs and their FGFRs.

TABLE 1 FGFR Variants Variant FGFR1 FGFR2 FGFR3 IIIb IIIc IIIb IIIc IIIbIIIc FGFR4 FGFR5 Ligands FGF-1 FGF-1 FGF-1 FGF-1 FGF-1 FGF-1 FGF-1 FGF-1FGF-2 FGF-2 FGF-3 FGF-2 FGF-9 FGF-2 FGF-2 FGF-2 FGF-3 FGF-4 FGF-7 FGF-4FGF-4 FGF-4 FGF-8 FGF-5 FGF-10 FGF-5 FGF-8 FGF-6 FGF-10 FGF-6 FGF-6FGF-9 FGF-8 FGF-8 FGF-8 FGF-9 FGF-17 FGF-9 FGF-17 Tissues Brain, bone,Brain, kidney, Brain, CNS, Lung, liver, Brain, skin, kidney, skin, skin,lung, liver, kidney, skin, kidney lung, testis lung, heart, glial cellslung, testis muscle, neuron Table 1 - FGFR variants and ligandaffinities. FGFR variants, associated ligands, and tissue distribution,see, e.g., . Powers et al, supra, (2000); and Reuss & von Bohlen undHalbach, supra, (2003).

Diversity in FGF signaling beyond the five receptors is achieved in partby the generation of alternatively spliced variants encoding distinctreceptor isoforms, see, e.g., Bernhard Reuss & Oliver von Bohlen undHalbach, Fibroblast growth factors and their receptors in the centralnervous system, 313(2) Cell Tissue Res. 139-157 (2003). The proteinregion that appears to have the highest influence on ligand bindingspecificity is a portion of the IgIII domain, for which isoforms encodedby three different splice variants have been identified. These threeisoforms, designated IgIIIa, IgIIIb and IgIIIc, have relative bindingaffinities for different FGFR family members. Alternative splicing inthe FGFR ligand binding domain, designated a and b, generates additionalreceptor isoforms with novel ligand affinities. Isoforms for IgIIIa,IgIIIb and IgIIIc have been identified for both FGFR1 and FGFR2. Thusfar, the IgIIIa isoform of FGFR3 and the IgIIIa and IgIIIb isoforms ofFGFR4 and FGFR5 have not been reported.

As mentioned above, FGFR3 commonly exists in two isoforms, FGFR3IIIc andFGFR3IIIb, which arise following alternative splicing of the primarytranscript in which either exon 8 or 9 respectively is skipped (see FIG.2). However, additional isoforms exist. For example, an FGFR3 isoformhas been described which lacks the acid box, see, e.g., Akio Shimizu etal, A novel alternatively spliced fibroblast growth factor receptor 3isoform lacking the acid box domain is expressed during chondrogenicdifferentiation of ATDC5 cells, 276(14) J. Biol. Chem. 11031-11040(2001). In another example, a novel, potentially cytoplasmic isoform wasrecently identified, called FGFR3S, in which exons 8, 9 and 10 arespliced out creating a FGFR3 that lacks the second half of IgIIIc andthe transmembrane domain, see, e.g., L-M. Sturla et al., FGFR3IIIS: anovel soluble FGFR3 spliced variant that modulates growth is frequentlyexpressed in tumour cells, 89(7) Br. J. Cancer 1276-1284 (2003).

Aspects of the present invention provide, in part, a method of detectingBoNT/A activity by contacting a sample to a cell that contains anexogenous FGFR3 wherein said contacted cell is capable of BoNT/Aintoxication and detecting the presence of BoNT/A activity of saidcontacted cell relative to a control cell, where a difference in saidBoNT/A activity of said contacted cell as compared to said control cellis indicative of BoNT/A activity. In an embodiment, a method ofdetecting BoNT/A activity comprises contacting a sample to a cell thattransiently contains an exogenous FGFR3 wherein said contacted cell iscapable of BoNT/A intoxication and detecting the presence of BoNT/Aactivity of said contacted cell relative to a control cell, where adifference in said BoNT/A activity of said contacted cell as compared tosaid control cell is indicative of BoNT/A activity. In anotherembodiment a method of detecting BoNT/A activity comprises contacting asample to a cell that stably contains an exogenous FGFR3 wherein saidcontacted cell is capable of BoNT/A intoxication and detecting thepresence of BoNT/A activity of said contacted cell relative to a controlcell, where a difference in said BoNT/A activity of said contacted cellas compared to said control cell is indicative of BoNT/A activity.

As used herein “botulinum toxin serotype A” is synonymous with “BoNT/A,”“type A,” or similar terminology referring unambiguously to Clostridiumbotulinum neurotoxin type A, means any of a number of polypeptideneurotoxins, and derivatives thereof, which can be purified fromClostridium botulinum serotype A strains and which share FGFR3 as a cellsurface receptor. Such neurotoxins include those found in orcorresponding to the following strains and accession numbers listed inTable 2.

TABLE 2 Strain Accession No. CL138 AAQ16535 137 AAQ16534 129 AAQ16533 13AAQ16532 42N AAQ16531 Hall A-hyper AAM75961 667Ab CAA61124 NCTC 2916CAA36289 Allergan-Hall A AAQ06331 62A AAA23262 Kyoto-F CAA51824 type ANIH BAA11051 NCTC 7272 7I03-H Kumgo AAO21363

As used herein, the term “Fibroblast Growth Factor 3 Receptor” issynonymous with “FGFR3” and means a FGFR3 peptide or peptidomimeticwhich binds BoNT/A in a manner that elicits a BoNT/A intoxicationresponse. FGFR3s useful in the invention encompass, without limitation,wild type FGFR3s, naturally occurring FGFR3 variants, non-naturallyFGFR3 variants, such as, e.g., genetically engineered variants producedby random mutagenesis or rational designed, and active fragments derivedfrom a FGFR3s. As a non-limiting example, a human FGFR3, naturallyoccurring human FGFR3 variants, non-naturally human FGFR3 variants, andhuman FGFR3 fragments that retain the ability to selectively bind BoNT/Aand mediate the intoxication process, can be useful as a BoNT/A receptorin aspects of the present invention. In another non-limiting example, abovine FGFR3, naturally occurring bovine FGFR3 variants, non-naturallybovine FGFR3 variants, and bovine FGFR3 fragments that retain theability to selectively bind BoNT/A and mediate the intoxication process,can be useful as a BoNT/A receptor in aspects of the present invention.In another non-limiting example, a rat FGFR3, naturally occurring ratFGFR3 variants, non-naturally rat FGFR3 variants, and rat FGFR3fragments that retain the ability to selectively bind BoNT/A and mediatethe intoxication process, can be useful as a BoNT/A receptor in aspectsof the present invention. In still another non-limiting example, a mouseFGFR3, naturally occurring mouse FGFR3 variants, non-naturally mouseFGFR3 variants, and mouse FGFR3 fragments that retain the ability toselectively bind BoNT/A and mediate the intoxication process, can beuseful as a BoNT/A receptor in aspects of the present invention. Inanother non-limiting example, a chicken FGFR3, naturally occurringchicken FGFR3 variants, non-naturally chicken FGFR3 variants, andchicken FGFR3 fragments that retain the ability to selectively bindBoNT/A and mediate the intoxication process, can be useful as a BoNT/Areceptor in aspects of the present invention. In another non-limitingexample, a frog FGFR3, naturally occurring frog FGFR3 variants,non-naturally frog FGFR3 variants, and frog FGFR3 fragments that retainthe ability to selectively bind BoNT/A and mediate the intoxicationprocess, can be useful as a BoNT/A receptor in aspects of the presentinvention. In another non-limiting example, a newt FGFR3, naturallyoccurring newt FGFR3 variants, non-naturally newt FGFR3 variants, andnewt FGFR3 fragments that retain the ability to selectively bind BoNT/Aand mediate the intoxication process, can be useful as a BoNT/A receptorin aspects of the present invention. In another non-limiting example, azebrafish FGFR3, naturally occurring zebrafish FGFR3 variants,non-naturally zebrafish FGFR3 variants, and zebrafish FGFR3 fragmentsthat retain the ability to selectively bind BoNT/A and mediate theintoxication process, can be useful as a BoNT/A receptor in aspects ofthe present invention. In is also understood that both nucleic acidmolecules, such as, e.g., DNA and RNA, that encode a FGFR3 disclosed inthe present specification and peptide molecules or peptidomimeticscomprising a FGFR3 disclosed in the present specification are useful inaspects of the present invention. SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25 and 27 disclose nucleic acid molecules encodingrepresentative of FGFR3s useful in aspects on the present invention,while SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 28disclose peptide molecules representative of FGFR3s useful in aspects onthe present invention.

As used herein, the term “peptidomimetic” is used broadly to mean apeptide-like molecule that selectively binds BoNT/A as the peptideBoNT/A receptor upon which it is structurally based. Suchpeptidomimetics include chemically modified peptides, peptide-likemolecules containing non-naturally occurring amino acids, and peptoids,which are peptide-like molecules resulting from oligomeric assembly ofN-substituted glycines, and selectively bind BoNT/A as the peptidesubstrate upon which the peptidomimetic is derived, see, e.g., Goodmanand Ro, Peptidomimetics for Drug Design, in “Burger's MedicinalChemistry and Drug Discovery” Vol. 1 (ed. M. E. Wolff; John Wiley & Sons1995), pages 803-861).

A variety of peptidomimetics are known in the art including, forexample, peptide-like molecules which contain a constrained amino acid,a non-peptide component that mimics peptide secondary structure, or anamide bond isostere. A peptidomimetic that contains a constrained,non-naturally occurring amino acid can include, for example, anα-methylated amino acid; an α,α-dialkyl-glycine or α-aminocycloalkanecarboxylic acid; an N^(α)-C^(α) cyclized amino acid; an N^(α)-methylatedamino acid; a β- or γ-amino cycloalkane carboxylic acid; anα,β-unsaturated amino acid; a β,β-dimethyl or β-methyl amino acid;β-substituted-2,3-methano amino acid; an NC^(δ) or C^(α)-C^(δ) cyclizedamino acid; or a substituted proline or another amino acid mimetic. Inaddition, a peptidomimetic which mimics peptide secondary structure cancontain, for example, a nonpeptidic β-turn mimic; γ-turn mimic; mimic ofβ-sheet structure; or mimic of helical structure, each of which is wellknown in the art. A peptidomimetic also can be a peptide-like moleculewhich contains, for example, an amide bond isostere such as aretro-inverso modification; reduced amide bond; methylenethioether ormethylenesulfoxide bond; methylene ether bond; ethylene bond; thioamidebond; trans-olefin or fluoroolefin bond; 1,5-disubstituted tetrazolering; ketomethylene or fluoroketomethylene bond or another amideisostere. One skilled in the art understands that these and otherpeptidomimetics are encompassed within the meaning of the term“peptidomimetic” as used herein.

Thus, in aspects of this embodiment, the FGFR3 can be a human FGFR3IIIbthat selectively binds BoNT/A which has, e.g., at least 70% amino acididentity with the FGFR3 of SEQ ID NO: 2, at least 75% amino acididentity with the FGFR3 of SEQ ID NO: 2, at least 80% amino acididentity with the FGFR3 of SEQ ID NO: 2, at least 85% amino acididentity with the FGFR3 of SEQ ID NO: 2, at least 90% amino acididentity with the FGFR3 of SEQ ID NO: 2 or at least 95% amino acididentity with the FGFR3 of SEQ ID NO: 2. In other aspects of thisembodiment, the FGFR3 is a human FGFR3IIIb that that selectively bindsBoNT/A which has, e.g., at most one, two, three, four, five, six, seven,eight, nine, or ten amino acid substitutions relative to the FGFR3 ofSEQ ID NO: 2.

In other aspects of this embodiment, the FGFR3 can be a human FGFR3IIIcthat selectively binds BoNT/A which has, e.g., at least 70% amino acididentity with the FGFR3 of SEQ ID NO: 4, at least 75% amino acididentity with the FGFR3 of SEQ ID NO: 4, at least 80% amino acididentity with the FGFR3 of SEQ ID NO: 4, at least 85% amino acididentity with the FGFR3 of SEQ ID NO: 4, at least 90% amino acididentity with the FGFR3 of SEQ ID NO: 4 or at least 95% amino acididentity with the FGFR3 of SEQ ID NO: 4. In other aspects of thisembodiment, the FGFR3 is a human FGFR3IIIc that that selectively bindsBoNT/A which has, e.g., at most one, two, three, four, five, six, seven,eight, nine, or ten amino acid substitutions relative to the FGFR3 ofSEQ ID NO: 4.

In other aspects of this embodiment, the FGFR3 can be a human FGFR3IIISthat selectively binds BoNT/A which has, e.g., at least 70% amino acididentity with the FGFR3 of SEQ ID NO: 6, at least 75% amino acididentity with the FGFR3 of SEQ ID NO: 6, at least 80% amino acididentity with the FGFR3 of SEQ ID NO: 6, at least 85% amino acididentity with the FGFR3 of SEQ ID NO: 6, at least 90% amino acididentity with the FGFR3 of SEQ ID NO: 6 or at least 95% amino acididentity with the FGFR3 of SEQ ID NO: 6. In other aspects of thisembodiment, the FGFR3 is a human FGFR3IIIS that that selectively bindsBoNT/A which has, e.g., at most one, two, three, four, five, six, seven,eight, nine, or ten amino acid substitutions relative to the FGFR3 ofSEQ ID NO: 6.

In other aspects of this embodiment, the FGFR3 can be a bovine FGFR3IIIcthat selectively binds BoNT/A which has, e.g., at least 70% amino acididentity with the FGFR3 of SEQ ID NO: 8, at least 75% amino acididentity with the FGFR3 of SEQ ID NO: 8, at least 80% amino acididentity with the FGFR3 of SEQ ID NO: 8, at least 85% amino acididentity with the FGFR3 of SEQ ID NO: 8, at least 90% amino acididentity with the FGFR3 of SEQ ID NO: 8 or at least 95% amino acididentity with the FGFR3 of SEQ ID NO: 8. In other aspects of thisembodiment, the FGFR3 is a bovine FGFR3IIIc that that selectively bindsBoNT/A which has, e.g., at most one, two, three, four, five, six, seven,eight, nine, or ten amino acid substitutions relative to the FGFR3 ofSEQ ID NO: 8.

In other aspects of this embodiment, the FGFR3 can be a mouse FGFR3IIIbthat selectively binds BoNT/A which has, e.g., at least 70% amino acididentity with the FGFR3 of SEQ ID NO: 10, at least 75% amino acididentity with the FGFR3 of SEQ ID NO: 10, at least 80% amino acididentity with the FGFR3 of SEQ ID NO: 10, at least 85% amino acididentity with the FGFR3 of SEQ ID NO: 10, at least 90% amino acididentity with the FGFR3 of SEQ ID NO: 10 or at least 95% amino acididentity with the FGFR3 of SEQ ID NO: 10. In other aspects of thisembodiment, the FGFR3 is a mouse FGFR3IIIc that that selectively bindsBoNT/A which has, e.g., at most one, two, three, four, five, six, seven,eight, nine, or ten amino acid substitutions relative to the FGFR3 ofSEQ ID NO: 10.

In other aspects of this embodiment, the FGFR3 can be a mouse FGFR3IIIcthat selectively binds BoNT/A which has, e.g., at least 70% amino acididentity with the FGFR3 of SEQ ID NO: 12, at least 75% amino acididentity with the FGFR3 of SEQ ID NO: 12, at least 80% amino acididentity with the FGFR3 of SEQ ID NO: 12, at least 85% amino acididentity with the FGFR3 of SEQ ID NO: 12, at least 90% amino acididentity with the FGFR3 of SEQ ID NO: 12 or at least 95% amino acididentity with the FGFR3 of SEQ ID NO: 12. In other aspects of thisembodiment, the FGFR3 is a mouse FGFR3IIIc that that selectively bindsBoNT/A which has, e.g., at most one, two, three, four, five, six, seven,eight, nine, or ten amino acid substitutions relative to the FGFR3 ofSEQ ID NO: 12.

In other aspects of this embodiment, the FGFR3 can be a mouseFGFR3-delAcid that selectively binds BoNT/A which has, e.g., at least70% amino acid identity with the FGFR3 of SEQ ID NO: 14, at least 75%amino acid identity with the FGFR3 of SEQ ID NO: 14, at least 80% aminoacid identity with the FGFR3 of SEQ ID NO: 14, at least 85% amino acididentity with the FGFR3 of SEQ ID NO: 14, at least 90% amino acididentity with the FGFR3 of SEQ ID NO: 14 or at least 95% amino acididentity with the FGFR3 of SEQ ID NO: 14. In other aspects of thisembodiment, the FGFR3 is a mouse FGFR3-delAcid that that selectivelybinds BoNT/A which has, e.g., at most one, two, three, four, five, six,seven, eight, nine, or ten amino acid substitutions relative to theFGFR3 of SEQ ID NO: 14.

In other aspects of this embodiment, the FGFR3 can be a rat FGFR3IIIbthat selectively binds BoNT/A which has, e.g., at least 70% amino acididentity with the FGFR3 of SEQ ID NO: 16, at least 75% amino acididentity with the FGFR3 of SEQ ID NO: 16, at least 80% amino acididentity with the FGFR3 of SEQ ID NO: 16, at least 85% amino acididentity with the FGFR3 of SEQ ID NO: 16, at least 90% amino acididentity with the FGFR3 of SEQ ID NO: 16 or at least 95% amino acididentity with the FGFR3 of SEQ ID NO: 16. In other aspects of thisembodiment, the FGFR3 is a rat FGFR3IIIb that that selectively bindsBoNT/A which has, e.g., at most one, two, three, four, five, six, seven,eight, nine, or ten amino acid substitutions relative to the FGFR3 ofSEQ ID NO: 16.

In other aspects of this embodiment, the FGFR3 can be a rat FGFR3IIIcthat selectively binds BoNT/A which has, e.g., at least 70% amino acididentity with the FGFR3 of SEQ ID NO: 18, at least 75% amino acididentity with the FGFR3 of SEQ ID NO: 18, at least 80% amino acididentity with the FGFR3 of SEQ ID NO: 18, at least 85% amino acididentity with the FGFR3 of SEQ ID NO: 18, at least 90% amino acididentity with the FGFR3 of SEQ ID NO: 18 or at least 95% amino acididentity with the FGFR3 of SEQ ID NO: 18. In other aspects of thisembodiment, the FGFR3 is a rat FGFR3IIIc that that selectively bindsBoNT/A which has, e.g., at most one, two, three, four, five, six, seven,eight, nine, or ten amino acid substitutions relative to the FGFR3 ofSEQ ID NO: 18.

In other aspects of this embodiment, the FGFR3 can be a chicken FGFR3that selectively binds BoNT/A which has, e.g., at least 70% amino acididentity with the FGFR3 of SEQ ID NO: 20, at least 75% amino acididentity with the FGFR3 of SEQ ID NO: 20, at least 80% amino acididentity with the FGFR3 of SEQ ID NO: 20, at least 85% amino acididentity with the FGFR3 of SEQ ID NO: 20, at least 90% amino acididentity with the FGFR3 of SEQ ID NO: 20 or at least 95% amino acididentity with the FGFR3 of SEQ ID NO: 20. In other aspects of thisembodiment, the FGFR3 is a chicken FGFR3 that that selectively bindsBoNT/A which has, e.g., at most one, two, three, four, five, six, seven,eight, nine, or ten amino acid substitutions relative to the FGFR3 ofSEQ ID NO: 20.

In other aspects of this embodiment, the FGFR3 can be a frog FGFR3-1that selectively binds BoNT/A which has, e.g., at least 70% amino acididentity with the FGFR3 of SEQ ID NO: 22, at least 75% amino acididentity with the FGFR3 of SEQ ID NO: 22, at least 80% amino acididentity with the FGFR3 of SEQ ID NO: 22, at least 85% amino acididentity with the FGFR3 of SEQ ID NO: 22, at least 90% amino acididentity with the FGFR3 of SEQ ID NO: 22 or at least 95% amino acididentity with the FGFR3 of SEQ ID NO: 22. In other aspects of thisembodiment, the FGFR3 is a frog FGFR3 that that selectively binds BoNT/Awhich has, e.g., at most one, two, three, four, five, six, seven, eight,nine, or ten amino acid substitutions relative to the FGFR3 of SEQ IDNO: 22.

In other aspects of this embodiment, the FGFR3 can be a frog FGFR3-2that selectively binds BoNT/A which has, e.g., at least 70% amino acididentity with the FGFR3 of SEQ ID NO: 24, at least 75% amino acididentity with the FGFR3 of SEQ ID NO: 24, at least 80% amino acididentity with the FGFR3 of SEQ ID NO: 24, at least 85% amino acididentity with the FGFR3 of SEQ ID NO: 24, at least 90% amino acididentity with the FGFR3 of SEQ ID NO: 24 or at least 95% amino acididentity with the FGFR3 of SEQ ID NO: 24. In other aspects of thisembodiment, the FGFR3 is a frog FGFR3 that that selectively binds BoNT/Awhich has, e.g., at most one, two, three, four, five, six, seven, eight,nine, or ten amino acid substitutions relative to the FGFR3 of SEQ IDNO: 24.

In other aspects of this embodiment, the FGFR3 can be a newt FGFR3 thatselectively binds BoNT/A which has, e.g., at least 70% amino acididentity with the FGFR3 of SEQ ID NO: 26, at least 75% amino acididentity with the FGFR3 of SEQ ID NO: 26, at least 80% amino acididentity with the FGFR3 of SEQ ID NO: 26, at least 85% amino acididentity with the FGFR3 of SEQ ID NO: 26, at least 90% amino acididentity with the FGFR3 of SEQ ID NO: 26 or at least 95% amino acididentity with the FGFR3 of SEQ ID NO: 26. In other aspects of thisembodiment, the FGFR3 is a newt FGFR3 that that selectively binds BoNT/Awhich has, e.g., at most one, two, three, four, five, six, seven, eight,nine, or ten amino acid substitutions relative to the FGFR3 of SEQ IDNO: 26.

In other aspects of this embodiment, the FGFR3 can be a zebrafish FGFR3that selectively binds BoNT/A which has, e.g., at least 70% amino acididentity with the FGFR3 of SEQ ID NO: 28, at least 75% amino acididentity with the FGFR3 of SEQ ID NO: 28, at least 80% amino acididentity with the FGFR3 of SEQ ID NO: 28, at least 85% amino acididentity with the FGFR3 of SEQ ID NO: 28, at least 90% amino acididentity with the FGFR3 of SEQ ID NO: 28 or at least 95% amino acididentity with the FGFR3 of SEQ ID NO: 28. In other aspects of thisembodiment, the FGFR3 is a zebrafish FGFR3 that that selectively bindsBoNT/A which has, e.g., at most one, two, three, four, five, six, seven,eight, nine, or ten amino acid substitutions relative to the FGFR3 ofSEQ ID NO: 28.

Other aspects of the present invention provide, in part, the optionaluse of a polysialogangliosides, especially those of the G1b series, suchas, e.g., GD1a, GD1b, GD3, GQ1b, or GT1b. Cell compositions comprising aFGFR3 and a polysialoganglioside can increase the selective binding ofBoNT/A relative to a composition not containing a polysialoganglioside.Thus, in an embodiment, a composition comprises a FGFR3 and optionally apolysialoganglioside. In aspects of this embodiment, a compositioncomprises a FGFR3 and optionally a G1b polysialoganglioside, such as,e.g., GD1a, GD1b, GD3, GQ1b, or GT1b.

Thus, in an embodiment, a method of detecting BoNT/A activity comprisescontacting a sample to a cell that contains an exogenous FGFR3 andoptionally a G1b polysialoganglioside wherein said contacted cell iscapable of BoNT/A intoxication and detecting the presence of BoNT/Aactivity of said contacted cell relative to a control cell, where adifference in said BoNT/A activity of said contacted cell as compared tosaid control cell is indicative of BoNT/A activity. In anotherembodiment, a method of detecting BoNT/A activity comprises contacting asample to a cell that transiently contains an exogenous FGFR3 and a G1bpolysialoganglioside wherein said contacted cell is capable of BoNT/Aintoxication and detecting the presence of BoNT/A activity of saidcontacted cell relative to a control cell, where a difference in saidBoNT/A activity of said contacted cell as compared to said control cellis indicative of BoNT/A activity. In another embodiment a method ofdetecting BoNT/A activity comprises contacting a sample to a cell thatstably contains an exogenous FGFR3 and a G1b polysialogangliosidewherein said contacted cell is capable of BoNT/A intoxication anddetecting the presence of BoNT/A activity of said contacted cellrelative to a control cell, where a difference in said BoNT/A activityof said contacted cell as compared to said control cell is indicative ofBoNT/A activity.

Other aspects of the present invention provide, in part, a method ofdetecting BoNT/A activity comprises contacting a sample to a cell thattransiently contains an exogenous FGFR3 wherein said contacted cell iscapable of BoNT/A intoxication and detecting the presence of BoNT/Aactivity of said contacted cell relative to a control cell, where adifference in said BoNT/A activity of said contacted cell as compared tosaid control cell is indicative of BoNT/A activity. As used herein, theterm “transiently containing” means a FGFR3 that is temporarilyintroduced into a cell in order to perform the assays disclosed in thepresent specification. Thus, aspects of a cell transiently containing aFGFR3 disclosed in the specification may include a cell that contains aFGFR3 for, e.g., at most about one day, at most about two days, at mostabout three days, at most about four days, at most about five days, andat most about six days, at most about seven days, at most about eightdays, at most about nine days and at most about ten days.

In an aspect of this embodiment, the FGFR3 can be encoded by the nucleicacid molecule from a mammalian FGFR3, such as, e.g., a human FGFR3, abovine FGFR3, a rat FGFR3 or a mouse FGFR3; a bird FGFR3, such as, e.g.,chicken FGFR3; an amphibian FGFR3, such as, e.g., a newt FGFR3 or a frogFGFR3; and a fish FGFR3, such as, e.g., a zebrafish FGFR3. In an aspectof this embodiment, a method of detecting BoNT/A activity comprisescontacting a sample to a cell that transiently contains a nucleic acidmolecule encoding an exogenous mammalian FGFR3 wherein said contactedcell is capable of BoNT/A intoxication and detecting the presence ofBoNT/A activity of said contacted cell relative to a control cell, wherea difference in said BoNT/A activity of said contacted cell as comparedto said control cell is indicative of BoNT/A activity. In another aspectof this embodiment, a method of detecting BoNT/A activity comprisescontacting a sample to a cell that transiently contains a nucleic acidmolecule encoding an exogenous bird FGFR3 wherein said contacted cell iscapable of BoNT/A intoxication and detecting the presence of BoNT/Aactivity of said contacted cell relative to a control cell, where adifference in said BoNT/A activity of said contacted cell as compared tosaid control cell is indicative of BoNT/A activity. In an aspect of thisembodiment, a method of detecting BoNT/A activity comprises contacting asample to a cell that transiently contains a nucleic acid moleculeencoding an exogenous amphibian FGFR3 wherein said contacted cell iscapable of BoNT/A intoxication and detecting the presence of BoNT/Aactivity of said contacted cell relative to a control cell, where adifference in said BoNT/A activity of said contacted cell as compared tosaid control cell is indicative of BoNT/A activity. In another aspect ofthis embodiment, a method of detecting BoNT/A activity comprisescontacting a sample to a cell that transiently contains a nucleic acidmolecule encoding an exogenous fish FGFR3 wherein said contacted cell iscapable of BoNT/A intoxication and detecting the presence of BoNT/Aactivity of said contacted cell relative to a control cell, where adifference in said BoNT/A activity of said contacted cell as compared tosaid control cell is indicative of BoNT/A activity.

In another aspect of this embodiment, the FGFR3 can be a mammalianFGFR3, such as, e.g., a human FGFR3, a bovine FGFR3, a rat FGFR3 or amouse FGFR3; a bird FGFR3, such as, e.g., chicken FGFR3; an amphibianFGFR3, such as, e.g., a newt FGFR3 or a frog FGFR3; and a fish FGFR3,such as, e.g., a zebrafish FGFR3. Thus in an embodiment, a method ofdetecting BoNT/A activity comprises contacting a sample to a cell thattransiently contains an exogenous FGFR3 wherein said contacted cell iscapable of BoNT/A intoxication and detecting the presence of BoNT/Aactivity of said contacted cell relative to a control cell, where adifference in said BoNT/A activity of said contacted cell as compared tosaid control cell is indicative of BoNT/A activity. In aspect of thisembodiment, the FGFR3 can be a mammalian FGFR3, such as, e.g., a humanFGFR3, a bovine FGFR3, a rat FGFR3 or a mouse FGFR3; a bird FGFR3, suchas, e.g., chicken FGFR3; an amphibian FGFR3, such as, e.g., a newt FGFR3or a frog FGFR3; and a fish FGFR3, such as, e.g., a zebrafish FGFR3. Inan aspect of this embodiment, a method of detecting BoNT/A activitycomprises contacting a sample to a cell that transiently contains anexogenous mammalian FGFR3 wherein said contacted cell is capable ofBoNT/A intoxication and detecting the presence of BoNT/A activity ofsaid contacted cell relative to a control cell, where a difference insaid BoNT/A activity of said contacted cell as compared to said controlcell is indicative of BoNT/A activity. In another aspect of thisembodiment, a method of detecting BoNT/A activity comprises contacting asample to a cell that transiently contains an exogenous bird FGFR3wherein said contacted cell is capable of BoNT/A intoxication anddetecting the presence of BoNT/A activity of said contacted cellrelative to a control cell, where a difference in said BoNT/A activityof said contacted cell as compared to said control cell is indicative ofBoNT/A activity. In an aspect of this embodiment, a method of detectingBoNT/A activity comprises contacting a sample to a cell that transientlycontains an exogenous amphibian FGFR3 wherein said contacted cell iscapable of BoNT/A intoxication and detecting the presence of BoNT/Aactivity of said contacted cell relative to a control cell, where adifference in said BoNT/A activity of said contacted cell as compared tosaid control cell is indicative of BoNT/A activity. In another aspect ofthis embodiment, a method of detecting BoNT/A activity comprisescontacting a sample to a cell that transiently contains an exogenousfish FGFR3 wherein said contacted cell is capable of BoNT/A intoxicationand detecting the presence of BoNT/A activity of said contacted cellrelative to a control cell, where a difference in said BoNT/A activityof said contacted cell as compared to said control cell is indicative ofBoNT/A activity.

Other aspects of the present invention provide, in part, a method ofdetecting BoNT/A activity comprises contacting a sample to a cell thatstably contains an exogenous FGFR3 wherein said contacted cell iscapable of BoNT/A intoxication and detecting the presence of BoNT/Aactivity of said contacted cell relative to a control cell, where adifference in said BoNT/A activity of said contacted cell as compared tosaid control cell is indicative of BoNT/A activity. As used herein, theterm “stably containing” means a FGFR3 that is introduced into a celland maintained for long periods of time in order to perform the assaysof the present specification. Stably-maintained nucleic acid moleculesencompass stably-maintained nucleic acid molecules that areextra-chromosomal and replicate autonomously and stably-maintainednucleic acid molecules that are integrated into the chromosomal materialof the cell and replicate non-autonomously. Thus aspects of a cellstably containing a FGFR3 disclosed in the specification may include acell that contains a FGFR3 for, e.g., at least ten days, at least 20 twodays, at least 30 days, at least forty days, at least 50 days, and atleast 60 days, at least 70 days, at least 80 days, at least 90 days andat least 100 days. Other aspects of a cell stably containing a FGFR3disclosed in the specification may include a cell that contains a FGFR3for, e.g., at least 100 days, at least 200 days, at least 300 days, atleast 400 days, and at least 500 days. Still other aspects of a cellstably containing a FGFR3 disclosed in the specification may include acell that permanently contains a FGFR3.

In an aspect of this embodiment, the FGFR3 can be encoded by the nucleicacid molecule from a mammalian FGFR3, such as, e.g., a human FGFR3, abovine FGFR3, a rat FGFR3 or a mouse FGFR3; a bird FGFR3, such as, e.g.,chicken FGFR3; an amphibian FGFR3, such as, e.g., a newt FGFR3 or a frogFGFR3; and a fish FGFR3, such as, e.g., a zebrafish FGFR3. In an aspectof this embodiment, a method of detecting BoNT/A activity comprisescontacting a sample to a cell that stably contains a nucleic acidmolecule encoding an exogenous mammalian FGFR3 wherein said contactedcell is capable of BoNT/A intoxication and detecting the presence ofBoNT/A activity of said contacted cell relative to a control cell, wherea difference in said BoNT/A activity of said contacted cell as comparedto said control cell is indicative of BoNT/A activity. In another aspectof this embodiment, a method of detecting BoNT/A activity comprisescontacting a sample to a cell that stably contains a nucleic acidmolecule encoding an exogenous bird FGFR3 wherein said contacted cell iscapable of BoNT/A intoxication and detecting the presence of BoNT/Aactivity of said contacted cell relative to a control cell, where adifference in said BoNT/A activity of said contacted cell as compared tosaid control cell is indicative of BoNT/A activity. In an aspect of thisembodiment, a method of detecting BoNT/A activity comprises contacting asample to a cell that stably contains a nucleic acid molecule encodingan exogenous amphibian FGFR3 wherein said contacted cell is capable ofBoNT/A intoxication and detecting the presence of BoNT/A activity ofsaid contacted cell relative to a control cell, where a difference insaid BoNT/A activity of said contacted cell as compared to said controlcell is indicative of BoNT/A activity. In another aspect of thisembodiment, a method of detecting BoNT/A activity comprises contacting asample to a cell that stably contains a nucleic acid molecule encodingan exogenous fish FGFR3 wherein said contacted cell is capable of BoNT/Aintoxication and detecting the presence of BoNT/A activity of saidcontacted cell relative to a control cell, where a difference in saidBoNT/A activity of said contacted cell as compared to said control cellis indicative of BoNT/A activity.

In another aspect of this embodiment, the FGFR3 can be a mammalianFGFR3, such as, e.g., a human FGFR3, a bovine FGFR3, a rat FGFR3 or amouse FGFR3; a bird FGFR3, such as, e.g., chicken FGFR3; an amphibianFGFR3, such as, e.g., a newt FGFR3 or a frog FGFR3; and a fish FGFR3,such as, e.g., a zebrafish FGFR3. In an aspect of this embodiment, amethod of detecting BoNT/A activity comprises contacting a sample to acell that stably contains an exogenous mammalian FGFR3 wherein saidcontacted cell is capable of BoNT/A intoxication and detecting thepresence of BoNT/A activity of said contacted cell relative to a controlcell, where a difference in said BoNT/A activity of said contacted cellas compared to said control cell is indicative of BoNT/A activity. Inanother aspect of this embodiment, a method of detecting BoNT/A activitycomprises contacting a sample to a cell that stably contains anexogenous bird FGFR3 wherein said contacted cell is capable of BoNT/Aintoxication and detecting the presence of BoNT/A activity of saidcontacted cell relative to a control cell, where a difference in saidBoNT/A activity of said contacted cell as compared to said control cellis indicative of BoNT/A activity. In an aspect of this embodiment, amethod of detecting BoNT/A activity comprises contacting a sample to acell that stably contains an exogenous amphibian FGFR3 wherein saidcontacted cell is capable of BoNT/A intoxication and detecting thepresence of BoNT/A activity of said contacted cell relative to a controlcell, where a difference in said BoNT/A activity of said contacted cellas compared to said control cell is indicative of BoNT/A activity. Inanother aspect of this embodiment, a method of detecting BoNT/A activitycomprises contacting a sample to a cell that stably contains anexogenous fish FGFR3 wherein said contacted cell is capable of BoNT/Aintoxication and detecting the presence of BoNT/A activity of saidcontacted cell relative to a control cell, where a difference in saidBoNT/A activity of said contacted cell as compared to said control cellis indicative of BoNT/A activity.

As mentioned above, a nucleic acid molecule can be used to express aFGFR3 disclosed in the present specification. It is envisioned that anyand all methods for introducing a nucleic acid molecule into a cell canbe used. Methods useful for introducing a nucleic acid molecule into acell including, without limitation, calcium phosphate-mediated, DEAEdextran-mediated, lipid-mediated, polybrene-mediated,polylysine-mediated, viral-mediated, microinjection, protoplast fusion,biolistic, electroporation and conjugation to an antibody, gramacidin S,artificial viral envelope or other intracellular carrier such as TAT.,see, e.g., Introducing Cloned Genes into Cultured Mammalian Cells, pp.16.1-16.62 (Sambrook & Russell, eds., Molecular Cloning A LaboratoryManual, Vol. 3, 3^(rd) ed. 2001); Alessia Colosimo et al., Transfer andexpression of foreign genes in mammalian cells, 29(2) Biotechniques314-318, 320-322, 324 (2000); Philip Washbourne & A. KimberleyMcAllister, Techniques for gene transfer into neurons, 12(5) Curr. Opin.Neurobiol. 566-573 (2002); and Current Protocols in Molecular Biology,John Wiley and Sons, pp 9.16.4-9.16.11 (2000). One skilled in the artunderstands that selection of a specific method to introduce a nucleicacid molecule into a cell will depend, in part, on whether the cell willtransiently contain a BoNT/A receptor or whether the cell will stablycontain a BoNT/A receptor.

As mentioned above, a FGFR3 disclosed in the present specification canbe introduced into a cell. It is envisioned that any and all methodsusing a delivery agent to introduce a FGFR3 into a cell can be used. Asused herein, the term “delivery agent” means any molecule that enablesor enhances internalization of a covalently-linked,non-covalently-linked or in any other manner associated with a FGFR3into a cell. Thus, the term “delivery agent” encompasses, withoutlimitation, proteins, peptides, peptidomimetics, small molecules,nucleic acid molecules, liposomes, lipids, viruses, retroviruses andcells that, without limitation, transport a covalently or non-covalentlylinked substrate to the cell membrane, cell cytoplasm or nucleus. Itfurther is understood that the term “delivery agent” encompassesmolecules that are internalized by any mechanism, including deliveryagents which function via receptor mediated endocytosis and those whichare independent of receptor mediated endocytosis.

A delivery agent useful in the invention also can be an agent thatenables or enhances cellular uptake of a covalently linked FGFR3, suchas, e.g., by chemical conjugation or by genetically produced fusionproteins. Methods that covalently link delivery agents and methods ofusing such agents are described in, e.g., Steven F. Dowdy, ProteinTransduction System and Methods of Use Thereof, InternationalPublication No WO 00/34308 (Jun. 15, 2000); Gerard Chassaing & AlainProchiantz, Peptides which can be Used as Vectors for the IntracellularAddresing of Active Molecuels, U.S. Pat. No. 6,080,724 (Jun. 27, 2000);Alan Frankel et al., Fusion Protein Comprising TAT-derived TransportMoiert, U.S. Pat. No. 5,674,980 (Oct. 7, 1995); Alan Frankel et al.,TAT-derived Transport Polypeptide Conjugates, U.S. Pat. No. 5,747,641(May 5, 1998); Alan Frankel et al., TAT-derived Transport Polypeptidesand Fusion Proteins, U.S. Pat. No. 5,804,604 (Sep. 8, 1998); Peter F. J.O'Hare et al., Use of Transport Proteins, U.S. Pat. No. 6,734,167 (May11, 2004); Yao-Zhong Lin & Jack J. Hawiger, Method for importingbiologically active molecules into cells, U.S. Pat. No. 5,807,746 (Sep.15, 1998); Yao-Zhong Lin & Jack J. Hawiger, Method for importingbiologically active molecules into cells, U.S. Pat. No. 6,043,339 (Mar.28, 2000); Yao-Zhong Lin et al., Sequence and Method for GeneticEngineering of Proteins with Cell Membrane Translocating Activity, U.S.Pat. No. 6,248,558 (Jun. 19, 2001); Yao-Zhong Lin et al., Sequence andMethod for Genetic Engineering of Proteins with Cell MembraneTranslocating Activity, U.S. Pat. No. 6,432,680 (Aug. 13, 2002); Jack J.Hawiger et al., Method for importing biologically active molecules intocells, U.S. Pat. No. 6,495,518 (Dec. 17, 2002); Yao-Zhong Lin et al.,Sequence and Method for Genetic Engineering of Proteins with CellMembrane Translocating Activity, U.S. Pat. No. 6,780,843 (Aug. 24,2004); Jonathan B. Rothbard & Paul A Wender, Method and Composition forEnhancing Transport Across Biological Membranes, U.S. Pat. No. 6,306,993(Oct. 23, 2001); Jonathan B. Rothbard & Paul A Wender, Method andComposition for Enhancing Transport Across Biological Membranes, U.S.Pat. No. 6,495,663 (Dec. 17, 2002); and Pamela B. Davis et al., Fusionproteins for protein delivery, U.S. Pat. No. 6,287,817 (Sep. 11, 2001).

A delivery agent useful in the invention also can be an agent thatenables or enhances cellular uptake of a non-covalently associatedFGFR3. Methods that function in the absence of covalent linkage andmethods of using such agents are described in, e.g., Gilles Divita etal, Peptide-mediated Transfection Agents and Methods of Use, U.S. Pat.No. 6,841,535 (Jan. 11, 2005); Philip L Felgner and Olivier Zelphati,Intracellular Protein Delivery Compositions and Methods of Use, U.S.Patent Publication No. 2003/0008813); and Michael Karas IntracellularDelivery of Small Molecules, Proteins and Nucleic Acids, U.S. PatentPublication 2004/0209797 (Oct. 21, 2004). Such peptide delivery agentscan be prepared and used by standard methods and are commerciallyavailable, see, e.g. the Chariot™ Reagent (Active Motif, Carlsbad,Calif.); BioPORTER® Reagent (Gene Therapy Systems, Inc., San Diego,Calif.), BioTrek™ Protein Delivery Reagent (Stratagene, La Jolla,Calif.), and Pro-Ject™ Protein Transfection Reagent (PierceBiotechnology Inc., Rockford, Ill.).

As mentioned above, a cell can stably contain a FGFR3 disclosed in thepresent specification. Methods useful for making and using a cells thatstably contain an FGFR3 are described in, e.g., Elizabeth E. Plowrightet al., Ectopic expression of fibroblast growth factor receptor 3promotes myeloma cell proliferation and prevents apoptosis, 95(3) Blood992-998 (2000); TC, see, e.g., Hiroyuki Onose et al., Over-expression offibroblast growth factor receptor 3 in a human thyroid carcinoma cellline results in overgrowth of the confluent cultures, 140(2) Eur. J.Endocrinol. 169-173 (1999); M. Kana et al., Signal transduction pathwayof human fibroblast growth factor receptor 3. Identification of a novel66-kDa phosphoprotein, 272(10) J. Biol. Chem. 6621-6628 (1997); andJanet E. Henderson et al., Expression of FGFR3 with the G380Rachondroplasia mutation inhibits proliferation and maturation of CFK2chondrocytic cells, 15(1) J. Bone Miner. Res. 155-165 (2000).

Another aspect of the present invention provides, in part, an expressionconstruct that allow for expression of a nucleic acid molecule encodinga FGFR3 disclosed in the present specification. These expressionconstructs comprise an open reading frame encoding a FGFR3 disclosed inthe present specification, operably-linked to control sequences from anexpression vector useful for expressing a FGFR3 in a cell. The term“operably linked” as used herein, refers to any of a variety of cloningmethods that can ligate a nucleic acid molecule disclosed in the presentspecification into an expression vector such that a peptide encoded bythe composition is expressed when introduced into a cell.Well-established molecular biology techniques that may be necessary tomake an expression construct disclosed in the present specificationincluding, but not limited to, procedures involving polymerase chainreaction (PCR) amplification restriction enzyme reactions, agarose gelelectrophoresis, nucleic acid ligation, bacterial transformation,nucleic acid purification, nucleic acid sequencing are routineprocedures well within the scope of one skilled in the art and from theteaching herein. Non-limiting examples of specific protocols necessaryto make an expression construct are described in e.g., MOLECULAR CLONINGA LABORATORY MANUAL, supra, (2001); and CURRENT PROTOCOLS IN MOLECULARBIOLOGY (Frederick M. Ausubel et al., eds. John Wiley & Sons, 2004).These protocols are routine procedures well within the scope of oneskilled in the art and from the teaching herein.

A wide variety of expression vectors can be employed for expressing anopen reading frame encoding a FGFR3 and include without limitation,viral expression vectors, prokaryotic expression vectors and eukaryoticexpression vectors including yeast, insect and mammalian expressionvectors. Non-limiting examples of expression vectors, along withwell-established reagents and conditions for making and using anexpression construct from such expression vectors are readily availablefrom commercial vendors that include, without limitation, BDBiosciences-Clontech, Palo Alto, Calif.; BD Biosciences Pharmingen, SanDiego, Calif.; Invitrogen, Inc, Carlsbad, Calif.; EMDBiosciences-Novagen, Madison, Wis.; QIAGEN, Inc., Valencia, Calif.; andStratagene, La Jolla, Calif. The selection, making and use of anappropriate expression vector are routine procedures well within thescope of one skilled in the art and from the teachings herein.

It is envisioned that any of a variety of expression systems may beuseful for expressing construct compositions disclosed in the presentspecification. An expression system encompasses both cell-based systemsand cell-free expression systems. Cell-based systems include, withoutlimited, viral expression systems, prokaryotic expression systems, yeastexpression systems, baculoviral expression systems, insect expressionsystems and mammalian expression systems. Cell-free systems include,without limitation, wheat germ extracts, rabbit reticulocyte extractsand E. coli extracts. Expression using an expression system can includeany of a variety of characteristics including, without limitation,inducible expression, non-inducible expression, constitutive expression,viral-mediated expression, stably-integrated expression, and transientexpression. Expression systems that include well-characterized vectors,reagents, conditions and cells are well-established and are readilyavailable from commercial vendors that include, without limitation,Ambion, Inc. Austin, Tex.; BD Biosciences-Clontech, Palo Alto, Calif.;BD Biosciences Pharmingen, San Diego, Calif.; Invitrogen, Inc, Carlsbad,Calif.; QIAGEN, Inc., Valencia, Calif.; Roche Applied Science,Indianapolis, Ind.; and Stratagene, La Jolla, Calif. Non-limitingexamples on the selection and use of appropriate heterologous expressionsystems are described in e.g., PROTEIN EXPRESSION. A PRACTICAL APPROACH(S. J. Higgins and B. David Hames eds., Oxford University Press, 1999);Joseph M. Fernandez & James P. Hoeffler, GENE EXPRESSION SYSTEMS. USINGNATURE FOR THE ART OF EXPRESSION (Academic Press, 1999); and Meena Rai &Harish Padh, Expression Systems for Production of Heterologous Proteins,80(9) CURRENT SCIENCE 1121-1128, (2001). These protocols are routineprocedures well within the scope of one skilled in the art and from theteaching herein.

An expression construct comprising a nucleic acid molecule encoding aFGFR3 disclosed in the present specification can be operationally-linkedto a variety of regulatory elements that can positively or negativelymodulate, either directly or indirectly, the expression of a nucleicacid molecule, such as, e.g., constitutive, tissue-specific, inducibleor synthetic promoters and enhancers. Non-limiting examples ofconstitutive regulatory elements include, e.g., the cytomegalovirus(CMV), herpes simplex virus thymidine kinase (HSV TK), simian virus 40(SV40) early, 5′ long terminal repeat (LTR), elongation factor-1a(EF-1α) and polybiquitin (UbC) regulatory elements. Non-limitingexamples of inducible regulatory elements useful in aspects of thepresent invention include, e.g., chemical-inducible regulatory elementssuch as, without limitation, alcohol-regulated, tetracycline-regulated,steroid-regulated, metal-regulated and pathogenesis-related; andphysical-inducible regulatory elements such as, without limitation,temperature-regulated and light-regulated. Such inducible regulatoryelements can be prepared and used by standard methods and arecommercially available, including, without limitation,tetracycline-inducible and tetracycline-repressible elements such as,e.g., Tet-On™ and Tet-Off™ (BD Biosciences-Clontech, Palo Alto, Calif.)and the T-REx™ (Tetracycline-Regulated Expression) and Flp-In™ T-REx™systems (Invitrogen, Inc., Carlsbad, Calif.); ecdysone-inducibleregulatory elements such as, e.g., the Complete Control® InducibleMammalian Expression System (Stratagene, Inc., La Jolla, Calif.);isopropyl β-D-galactopyranoside (IPTG)-inducible regulatory elementssuch as, e.g., the LacSwitch® ^(II) Inducible Mammalian ExpressionSystem (Stratagene, Inc., La Jolla, Calif.); and steroid-inducibleregulatory elements such as, e.g., the chimeric progesterone receptorinducible system, GeneSwitch™ (Invitrogen, Inc., Carlsbad, Calif.). Theskilled person understands that these and a variety of otherconstitutive and inducible regulatory systems are commercially availableor well known in the art and can be useful in the invention forcontrolling expression of a nucleic acid molecule which encodes a BoNT/Areceptor.

In an embodiment, a nucleic acid molecule encoding a FGFR3 canoptionally be linked to a regulatory element such as a constitutiveregulatory element. In aspects of this embodiment, a nucleic acidmolecule encoding a mammalian FGFR3 can optionally be linked to aregulatory element such as a constitutive regulatory element; a nucleicacid molecule encoding a bird FGFR3 can optionally be linked to aregulatory element such as a constitutive regulatory element; a nucleicacid molecule encoding an amphibian FGFR3 can optionally be linked to aregulatory element such as a constitutive regulatory element; and anucleic acid molecule encoding a fish FGFR3 can optionally be linked toa regulatory element such as a constitutive regulatory element.

In another embodiment, a nucleic acid molecule encoding a FGFR3 canoptionally be linked to a regulatory element such as an inducibleregulatory element. In aspects of this embodiment, a nucleic acidmolecule encoding a mammalian FGFR3 can optionally be linked to aregulatory element such as a inducible regulatory element; a nucleicacid molecule encoding a bird FGFR3 can optionally be linked to aregulatory element such as a inducible regulatory element; a nucleicacid molecule encoding an amphibian FGFR3 can optionally be linked to aregulatory element such as a inducible regulatory element; and a nucleicacid molecule encoding a fish FGFR3 can optionally be linked to aregulatory element such as a inducible regulatory element. In anotheraspect of this embodiment, expression of the nucleic acid molecule isinduced using, e.g., tetracycline-inducible, ecdysone-inducible orsteroid-inducible.

It is understood that a FGFR3 useful in aspects of the present inventionoptionally can include one or more additional components. As anon-limiting example, a flexible spacer sequence such as poly-glycinesequences can be included in a FGFR3 useful in the invention. A usefulFGFR3 can further include, without limitation, one or more of thefollowing: epitope-binding tags, such as. e.g., FLAG, Express™, humanInfluenza virus hemaglutinin (HA), human p62^(c-MYc) protein (c-MYC),Vesicular Stomatitis Virus Glycoprotein (VSV-G), glycoprotein-Dprecursor of Herpes simplex virus (HSV), V5, and AU1; affinity-binding,such as, e.g., polyhistidine (HIS), streptavidin binding peptide(strep), and biotin or a biotinylation sequence; peptide-bindingregions, such as, e.g., the glutathione binding domain ofglutathione-S-transferase, the calmodulin binding domain of thecalmodulin binding protein, and the maltose binding domain of themaltose binding protein; immunoglobulin hinge region; anN-hydroxysuccinimide linker; a peptide or peptidomimetic hairpin turn;or a hydrophilic sequence or another component or sequence that, forexample, promotes the solubility or stability of a FGFR3. Non-limitingexamples of specific protocols for selecting, making and using anappropriate binding peptide are described in, e.g., Epitope Tagging, pp.17.90-17.93 (Sambrook and Russell, eds., Molecular Cloning A LaboratoryManual, Vol. 3, 3^(rd) ed. 2001); Antibodies: A Laboratory Manual(Edward Harlow & David Lane, eds., Cold Spring Harbor Laboratory Press,2^(nd) ed. 1998); and Using Antibodies: A Laboratory Manual: PortableProtocol No. I (Edward Harlow & David Lane, Cold Spring HarborLaboratory Press, 1998). In addition, non-limiting examples of bindingpeptides as well as well-characterized reagents, conditions andprotocols are readily available from commercial vendors that include,without limitation, BD Biosciences-Clontech, Palo Alto, Calif.; BDBiosciences Pharmingen, San Diego, Calif.; Invitrogen, Inc, Carlsbad,Calif.; QIAGEN, Inc., Valencia, Calif.; and Stratagene, La Jolla, Calif.These protocols are routine procedures well within the scope of oneskilled in the art and from the teaching herein.

Aspects of the present invention provide, in part, a cell that containsan exogenous FGFR3 wherein said cell is capable of BoNT/A intoxication.As used herein, the term “cell,” means any eukaryotic cell thatexpresses, or can be engineered to express, at least one exogenous FGFR3that binds BoNT/A. The term cell encompasses cells from a variety oforganisms, such as, e.g., murine, rat, porcine, bovine, equine, primateand human cells; from a variety of cell types such as, e.g., neural andnon-neural; and can be isolated from or part of a heterogeneous cellpopulation, tissue or organism. It is understood that cells useful inaspects of the invention can included, without limitation, primarycells; cultured cells; established cells; normal cells; transformedcells; tumor cells; infected cells; proliferating and terminallydifferentiated cells; and stably or transiently transfected cells,including stably and transiently transfected cells. It is furtherunderstood that cells useful in aspects of the invention can be in anystate such as proliferating or quiescent; intact or permeabilized suchas through chemical-mediated transfection such as, e.g., calciumphosphate-mediated, diethyl-laminoethyl (DEAE) dextran-mediated,lipid-mediated, polyethyleneimine (PEI)-mediated, polybrene-mediated,and protein delivery agents; physical-mediated transfection, such as,e.g., biolistic particle delivery, microinjection and electroporation;and viral-mediated transfection, such as, e.g., retroviral-mediatedtransfection. It is further understood that cells useful in aspects ofthe invention may include those which express a FGFR3 under control of aconstitutive, tissue-specific, cell-specific or inducible promoterelement, enhancer element or both.

As used herein, the term “cell capable of BoNT/A intoxication” means acell that can enable the overall cellular mechanism whereby BoNT/Aproteolytically cleaves a substrate, such as, e.g., SNAP-25, andencompasses the binding of BoNT/A to a low or high affinity receptor,the internalization of the toxin/receptor complex, the translocation ofthe BoNT/A light chain into the cytoplasm and the enzymatic targetmodification of a BoNT/A substrate. By definition, a cell capable ofBoNT/A intoxication must express a FGFR3. As a non-limiting example, aneuronal or non-neuronal cell can be transiently or stably engineered toexpress an exogenous nucleic acid molecule encoding a FGFR3. As anothernon-limiting example, a neuronal or non-neuronal cell can be transientlyengineered to contain an exogenous FGFR3.

Cells useful in aspects of the invention include both neuronal andnon-neuronal cells. Neuronal cells useful in aspects of the inventioninclude, without limitation, primary neuronal cells; immortalized orestablished neuronal cells; transformed neuronal cells; neuronal tumorcells; stably and transiently transfected neuronal cells and furtherinclude, yet are not limited to, mammalian, murine, rat, primate andhuman neuronal cells. Non-limiting examples of neuronal cells useful inaspects of the invention include, e.g., peripheral neuronal cells, suchas, e.g., motor neurons and sensory neurons; and CNS neuronal cells,such as, e.g., spinal cord neurons like embryonic spinal cord neurons,dorsal root ganglia (DRG) neurons, cerebral cortex neurons, cerebellarneurons, hippocampal neurons and motor neurons. Neuronal cells useful inthe invention can be, for example, central nervous system (CNS) neurons;neuroblastoma cells; motor neurons, hippocampal neurons or cerebellarneurons and further can be, without limitation, Neuro-2A, SH-SY5Y,NG108-15, N1E-115 or SK-N-DZ cells. The skilled person understands thatthese and additional primary and established neurons can be useful inthe cells and methods of the invention.

Neurons useful in aspects of the invention include, without limitation,primary cultures such as primary cultures of embryonic dorsal rootganglion (DRG) neurons. As one example, primary cultures of embryonicrat DRG neurons are described in Mary J. Welch et al., Sensitivity ofembryonic rat dorsal root ganglia neurons to Clostridium botulinumneurotoxins, 38(2) Toxicon 245 258 (2000); and primary cultures of fetalspinal cord neurons, for example, primary cultures of murine fetalspinal cord neurons are described in Elaine A. Neale et al., Botulinumneurotoxin A blocks synaptic vesicle exocytosis but not endocytosis atthe nerve terminal, 147(6) J. Cell Biol. 1249-1260 (1999), and John A.Chaddock et al., Inhibition of vesicular secretion in both neuronal andnon-neuronal cells by a retargeted endopeptidase derivative ofClostridium botulinum neurotoxin type A, 68(5) Infect. Immun. 2587-2593(2000). Thus, in an embodiment, a cell capable of BoNT/A intoxicationcan be a neuron that contains an exogenous FGFR3. In aspects of thisembodiment, a neuron can be a neuron from, e.g., a primary culture, anembryonic dorsal root ganglion primary culture or a fetal spinal cordprimary culture. As non-limiting examples, cells useful according to amethod disclosed in the present specification can include, a primaryneuronal cell that contains an exogenous FGFR3, such as, e.g., a ratembryonic dorsal root ganglion (DRG) neuron that contains an exogenousFGFR3 or a murine fetal spinal cord neuron that contains an exogenousFGFR3.

Neuronal cell lines useful in aspects of the invention include, withoutlimitation, neuroblastoma cell lines, neuronal hybrid cell lines, spinalcord cell lines, central nervous system cell lines, cerebral cortex celllines, dorsal root ganglion cell lines, hippocampal cell lines andpheochromocytoma cell lines.

Neuroblastoma cell lines, such as, e.g., murine, rat, primate or humanneuroblastoma cell lines can be useful in aspects of the invention.Neuroblastoma cell lines useful in aspects of the invention include,without limitation, BE(2)-C (ATCC CRL-2268; ECACC 95011817), BE(2)-M17(ATCC CRL-2267; ECACC 95011816), C1300 (ECACC 93120817), CHP-212 (ATCCCRL-2273), CHP-126 (DSMZ ACC 304), IMR 32 (ATCC CRL-127; ECACC 86041809;DSMZ ACC 165), KELLY (ECACC 92110411; DSMZ ACC 355), LA-N-2, see, e.g.,Robert C. Seeger et al., Morphology, growth, chromosomal pattern andfibrinolytic activity of two new human neuroblastoma cell lines, 37(5)Cancer Res. 1364-1371 (1977); and G. J. West et al., Adrenergic,cholinergic, and inactive human neuroblastoma cell lines with theaction-potential Na+ ionophore, 37(5) Cancer Res. 1372-1376 (1977),MC-IXC (ATCC CRL-2270), MHH-NB-11 (DSMZ ACC 157), N18Tg2 (DSMZ ACC 103),N1E-115 (ATCC CCL-2263; ECACC 88112303), N4TG3 (DSMZ ACC 101), Neuro-2A(ATCC CCL-131; ECACC 89121404; DSMZ ACC 148), NB41A3 (ATCC CCL-147;ECACC 89121405), NS20Y (DSMZ ACC 94), SH-SY5Y (ATCC CRL-2266; ECACC94030304; DSMZ ACC 209), SIMA (DSMZ ACC 164), SK-N-DZ (ATCC CRL-2149;ECACC 94092305), SK-N-F1 (ATCC CRL-2142, ECACC 94092304), SK-N-MC (ATCCHTB-10, DSMZ ACC 203) and SK-N-SH (ATCC HTB-11, ECACC 86012802). Thus,in an embodiment, a cell capable of BoNT/A intoxication can be aneuroblastoma cell that contains an exogenous FGFR3. In aspects of thisembodiment, a neuroblastoma cell can be, e.g., BE(2)-C, BE(2)-M17,C1300, CHP-212, CHP-126, IMR 32, KELLY, LA-N-2, MC-IXC, MHH-NB-11,N18Tg2, N1E-115, N4TG3, Neuro-2A, NB41A3, NS20Y, SH-SY5Y, SIMA, SK-N-DZ,SK-N-F1, SK-N-MC and SK-N-SH. As non-limiting examples, cells useful fordetecting BoNT/A activity according to a method disclosed in the presentspecification can include, a neuroblastoma cell that contains anexogenous FGFR3, such as, e.g., a SH-SY5Y cell that contains anexogenous FGFR3; a Neuro-2a cell that contains an exogenous FGFR3; and aN1E-115 cell that contains an exogenous FGFR3; and a SK-N-DZ cell thatcontains an exogenous FGFR3.

Neuronal hybrid cell lines, such as, e.g., murine, rat, primate andhuman hybrid neuronal cell lines can be useful in aspects of theinvention. Such hybrid cell lines include neuroblastoma/glioma hybrids,such as, e.g., N18 (ECACC 88112301), NG108-15 (ATCC HB-12317, ECACC88112302) and NG115-401L (ECACC 87032003); neuroblastoma/motor neuronhybrids, such as, e.g., NSC-19 and NSC-34, which express motor neuroncharacteristics, display a multipolar neuron-like phenotype, expresshigh levels of choline acetyltransferase (CHAT), generate actionpotentials, express neurofilament triplet proteins and synthesize, storeand release acetylcholine, see, e.g., N. R. Cashman et al.,Neuroblastoma x spinal cord (NSC) hybrid cell lines resemble developingmotor neurons, 194(3) Dev. Dyn. 209-221 (1992); and Christopher J.Eggett et al., Development and characterisation of a glutamate-sensitivemotor neuronal cell line, 74(5) J. Neurochem. 1895-1902 (2000);neuroblastoma/root ganglion neuron hybrids, such as, e.g., F11, see,e.g., Doros Platika et al., Neuronal traits of clonal cell lines derivedby fusion of dorsal root ganglia neurons with neuroblastoma cells,82(10) Proc. Natl. Acad. Sci. U.S.A. 3499-3503 (1985), ND-E (ECACC92090915), ND-U1 (ECACC 92090916), ND7/23 (ECACC 92090903), ND8/34(ECACC 92090904) and ND27 (ECACC 92090912); neuroblastoma/hippocampalneuron hybrids, such as, e.g., HN-33, see, e.g., Henry J. Lee et al.,Neuronal properties and trophic activities of immortalized hippocampalcells from embryonic and young adult mice. 10(6) J. Neurosci. 1779-1787(1990). Thus, in an embodiment, a cell capable of BoNT/A toxinintoxication can be a hybrid neuron that contains an exogenous FGFR3. Inaspects of this embodiment, a hybrid neuron can be, e.g., aneuroblastoma/glioma hybrid cell that contains an exogenous FGFR3, aneuroblastoma/motor neuron hybrid cell that contains an exogenous FGFR3,a neuroblastoma/root ganglion neuron hybrid cell that contains anexogenous FGFR3 and a neuroblastoma/hippocampal neuron hybrid cell thatcontains an exogenous FGFR3. In further aspects of this embodiment, aneuroblastoma/glioma hybrid can be, e.g., N18, NG108-15 and NG115-401L.In further aspects of this embodiment, a neuroblastoma/motor neuronhybrid can be, e.g., NSC-19 and NSC-32. In further aspects of thisembodiment, a neuroblastoma/root ganglion neuron hybrid can be, e.g.,F11, ND-E, ND-U1, ND7/23, ND8/34 and ND27. In further aspects of thisembodiment, a neuroblastoma/hippocampal neuron hybrid can be, e.g.,HN-33. As non-limiting examples, cells useful for detecting BoNT/Aactivity according to a method disclosed in the present specificationcan include, a neuronal hybrid cell, such as, e.g., a NG108-15 cell thatcontains an exogenous FGFR3.

Spinal cord cell lines, such as, e.g., murine, rat, primate or humanspinal cord cell lines can be useful in aspects of the invention andinclude, without limitation, TE 189.T (ATCC CRL-7947) and M4b, see,e.g., Ana M. Cardenas et al., Establishment and characterization ofimmortalized neuronal cell lines derived from the spinal cord of normaland trisomy 16 fetal mice, an animal model of Down syndrome, 68(1) J.Neurosci. Res. 46-58 (2002). As an example, a human spinal cord cellline can be generated from precursors of human embryonic spinal cordcells (first trimester embryos) that are immortalized with atetracycline repressible v-myc oncogene as described in Ronghao Li etal., Motorneuron differentiation of immortalized human spinal cord celllines, 59(3) J. Neurosci. Res. 342-352 (2000). Such cells can beexpanded indefinitely in proliferative growth conditions before rapiddifferentiation (4-7 days) into functional neurons that express neuronalphenotypic markers such as choline acetyltransferase. As anotherexample, a murine spinal cord cell line can be prepared by immortalizingan embryonic spinal cord culture using transforming media. Such a spinalcord cell line can be, for example, the murine M4b line and can expressneuronal markers such as NSE, synaptophysin, MAP 2 and cholineacetyltransferase, and can release acetylcholine upon appropriatestimulation, see, e.g., Cardenas et al., supra, (2002). Thus, in anembodiment, a cell capable of BoNT/A intoxication can be a spinal cordcell that contains an exogenous FGFR3. In aspects of this embodiment, aspinal cord cell that contains an exogenous FGFR3 can be, e.g., a TE189.T cell that contains an exogenous FGFR3 and a M4b cell that containsan exogenous FGFR3.

Central nervous system (CNS) cell lines, such as, e.g., murine, rat,primate and human CNS cell lines, can be useful in aspects of theinvention. A useful CNS cell line can be, for example, a human CNS cellline immortalized with a tetracycline repressible v-myc oncogene asdescribed in Dinah W. Sah et al., Bipotent progenitor cell lines fromthe human CNS, 15(6) Nat. Biotechnol. 574-580 (1997). Upon repression ofthe oncogene, the cells differentiate into neurons. Thus, in anembodiment, a cell capable of BoNT/A intoxication can be a CNS cell thatcontains an exogenous FGFR3.

Cerebral cortex cell lines, such as, e.g., murine, rat, primate andhuman cerebral cortex cell lines, can be useful in aspects of theinvention and include, without limitation, CNh, see, e.g., Ana M.Cardenas et al., Calcium signals in cell lines derived from the cerebralcortex of normal and trisomy 16 mice, 10(2) Neuroreport 363-369 (1999),HCN-1a (ATCC CRL-10442) and HCN-2 (ATCC CRL-10742). As an example,murine cortex primary cultures from 12-16 days embryos can beimmortalized, for example, by culturing the cells in conditioned mediafrom a rat thyroid cell line that induces transformation in vitro. Theimmortalized cells can be differentiated into neurons expressingneuronal markers using the appropriate media; these differentiated cellsexpress choline acetyltransferase and secrete acetylcholine andglutamate in response to depolarization and nicotine stimulation, see,e.g., David D. Allen et al., Impaired cholinergic function in cell linesderived from the cerebral cortex of normal and trisomy 16 mice, 12(9)Eur. J. Neurosci. 3259-3264 (2000). Thus, in an embodiment, a cellcapable of BoNT/A intoxication can be a cerebral cortex cell thatcontains an exogenous FGFR3. In aspects of this embodiment, a cerebralcortex cell that contains an exogenous FGFR3 can be, e.g., a CNh cellthat contains an exogenous FGFR3, HCN-1a cell that contains an exogenousFGFR3 and HCN-2 cell that contains an exogenous FGFR3.

Dorsal root ganglia cell lines, such as, e.g., murine, rat, primate andhuman dorsal root ganglia cell lines, can be useful in aspects of theinvention and include, without limitation, G4b, see, e.g., David D.Allen et al., A dorsal root ganglia cell line derived from trisomy 16fetal mice, a model for Down syndrome, 13(4) Neuroreport 491-496 (2002).Embryonic dorsal root ganglia primary cultures can be immortalized withtransforming conditioned media as described above. Upon differentiation,the cell line exhibits neuronal traits and lacks glial markers byimmunohistochemistry. Release of neurotransmitters such as acetylcholinecan be induced in response to potassium and nicotine, see, e.g., Allenet al., supra, (2002). Thus, in an embodiment, a cell capable of BoNT/Aintoxication can be a dorsal root ganglia cell that contains anexogenous FGFR3. In aspects of this embodiment, a dorsal root gangliacell can be, e.g., a G4b cell that contains an exogenous FGFR3.

Hippocampal cell lines, such as, e.g., murine, rat, primate and humanhippocampal lines can be useful in aspects of the invention and include,without limitation, HT-4, see, e.g., K. Frederiksen et al.,Immortalization of precursor cells from the mammalian CNS, 1(6) Neuron439-448 (1988) and HT-22, see, e.g., John B. Davis and Pamela Maher,Protein kinase C activation inhibits glutamate-induced cytotoxicity in aneuronal cell line, 652(1) Brain Res. 169-173 (1994). As a non-limitingexample, the murine hippocampal cell line HT-22 can be useful in theinvention. As a further non-limiting example, the immortalized HN33hippocampal cell line can be useful in the invention. This hippocampalcell line was derived from the fusion of primary neurons from thehippocampus of postnatal day 21 mice with the N18TG2 neuroblastoma cellline, and, when differentiated, shares membrane properties with adulthippocampal neurons in primary culture, see, e.g., Henry J. Lee et al.,Neuronal Properties and Trophic Activities of Immortalized HippocampalCells from Embryonic and Young Adult Mice, 19(6) J. Neurosci. 1779-1787(1990); and Henry J. Lee et al., Immortalized young adult neurons fromthe septal region: generation and characterization, 52(1-2) Brain Res.Dev Brain Res. 219-228 (1990). Thus, in an embodiment, a cell capable ofBoNT/A intoxication can be a hippocampal cell that contains an exogenousFGFR3. In aspects of this embodiment, a hippocampal cell that containsan exogenous FGFR3 can be, e.g., a HT-4 cell that contains an exogenousFGFR3, a HT-22 cell that contains an exogenous FGFR3 and a HN33 cellthat contains an exogenous FGFR3.

A variety of non-neuronal cells are useful in aspects of the invention.Non-neuronal cells useful in aspects of the invention include, withoutlimitation, primary non-neuronal cells; immortalized or establishednon-neuronal cells; transformed non-neuronal cells; non-neuronal tumorcells; stably and transiently transfected non-neuronal cells and furtherinclude, yet are not limited to, mammalian, murine, rat, primate andhuman non-neuronal cells. Non-neuronal cells useful in aspects of theinvention further include, without limitation, any of the followingprimary or established cells: anterior pituitary cells; adrenal cells,such as, e.g., chromaffin cells of the adrenal medulla; pancreaticcells, such as, e.g., pancreatic acinar cells, pancreatic islet β cellsand insulinoma HIT or INS-1 cells; ovarian cells, such as, e.g.,steroid-producing ovarian cells; kidney cells, such as, e.g., innermedullary collecting duct (IMCD) cells; stomach cells, such as, e.g.,enterochromaffin cells; blood cells, such as. e.g., eurythrocytes,leucocytes, platelets, neutrophils, eosinophils, mast cells; epithelialcells, such as. e.g., those of the apical plasma membrane; fibroblasts;thyroid cells; chondrocytes; muscle cells; hepatocytes; glandular cellssuch as, e.g., pituitary cells, adrenal cells, chromaffin cells; andcells involved in glucose transporter (GLUT4) translocation. Thus, in anembodiment, a cell capable of BoNT/A intoxication can be a non-neuronalcell. In aspects of this embodiment, a non-neuronal cell can be from aprimary or established non-neuronal cell line from the, e.g., anteriorpituitary cells, adrenal cells, pancreatic cells, ovarian cells, kidneycells, stomach cells, blood cells, epithelial cells, fibroblasts,thyroid cells, chondrocytes, muscle cells, hepatocytes and glandularcells.

As non-limiting examples, cells useful for detecting BoNT/A activityaccording to a method disclosed in the present specification caninclude, a primary or established non-neuronal cell that contains anexogenous FGFR3, such as, e.g., a chromaffin cell that contains anexogenous FGFR3 or pancreatic acinar cell that contains an exogenousFGFR3; a primary neuronal cell that contains an exogenous FGFR3.

As discussed above, cells useful in the invention include neuronal andnon-neuronal cells that express low or undetectable levels of endogenousreceptor but which have been transfected with, or otherwise engineeredto express, one or more exogenous nucleic acid molecules encoding one ormore FGFR3s. Cells useful in aspects of the present invention furtherinclude, without limitation, transformed, tumor or other cells whichover-express one or more exogenous FGFR3s. It is understood that theover-expressed receptor can be a wild type form of the receptor or caninclude one or more amino acid modifications as compared to the wildtype receptor, with the proviso that the process of BoNT/A intoxicationcan still occur. As a non-limiting example, cells useful for detectingBoNT/A activity encompass those which express or over-express anexogenous mammalian FGFR3, such as, e.g., a human FGFR3, a bovine FGFR3,a rat FGFR3 or a mouse FGFR3. As another non-limiting example, cellsuseful for detecting BoNT/A activity encompass those which express orover-express an exogenous bird FGFR3, such as, e.g., chicken FGFR3. Asanother non-limiting example, cells useful for detecting BoNT/A activityencompass those which express or over-express an exogenous amphibianFGFR3, such as, e.g., a newt FGFR3 or a frog FGFR3. As anothernon-limiting example, cells useful for detecting BoNT/A activityencompass those which express or over-express an exogenous fish FGFR3,such as, e.g., a zebrafish FGFR3.

Thus, in an embodiment, a cell capable of BoNT/A intoxication can be acell stably expressing an exogenous FGFR3. In aspects of thisembodiment, a cell capable of BoNT/A intoxication can be a cell stablyexpressing an exogenous mammalian FGFR3, such as, e.g., a human FGFR3, abovine FGFR3, a rat FGFR3 or a mouse FGFR3. In other aspects of thisembodiment, a cell capable of BoNT/A intoxication can be a cell stablyexpressing an exogenous bird FGFR3, such as, e.g., chicken FGFR3. Inother aspects of this embodiment, a cell capable of BoNT/A intoxicationcan be a cell stably expressing an exogenous amphibian FGFR3, such as,e.g., a newt FGFR3 or a frog FGFR3. In other aspects of this embodiment,a cell capable of BoNT/A intoxication can be a cell stably expressing anexogenous fish FGFR3, such as, e.g., a zebrafish FGFR3.

Aspects of the present invention provide, in part, detecting thepresence of BoNT/A activity of said contacted cell relative to a controlcell, where a difference in said BoNT/A activity of said contacted cellas compared to said control cell is indicative of BoNT/A activity. Asused herein, the term “control cell” means a cell of the same or similartype as the contacted cell and grown under the same conditions but whichis not contacted with any sample or is contacted with a defined negativesample or a defined positive sample. One skilled in the art understandsthat a variety of control cells are useful in the methods disclosed inthe present specification and that a control cell can be a positivecontrol cell or a negative control cell. A control cell can be, forexample, a negative control cell such as a similar or identical cellcontaining the same or similar FGFR3 that is contacted with a similar,defined negative sample, which is known to lack active BoNT/A, or thatis not contacted with any sample. A control cell also can be, forexample, a positive control cell such as a similar or identical cellcontaining the same or similar FGFR3 contacted with a defined positivesample, which is known to include active BoNT/A.

A wide variety of assays can be used to determine the presence of BoNT/Aactivity, including direct and indirect assays for toxin uptake. Assaysthat determine BoNT/A binding or uptake properties can be used to assessBoNT/A activity. Such assays include, without limitation, cross-linkingassays using labeled BoNT/A, such as, e.g., BoNT/A-SBED, see, e.g.,Example II of the present specification and [¹²⁵I] BoNT/A, see, e.g.,Noriko Yokosawa et al., Binding of Clostridium botulinum type Cneurotoxin to different neuroblastoma cell lines, 57(1) Infect. Immun.272-277 (1989); Noriko Yokosawa et al., Binding of botulinum type C1, Dand E neurotoxins to neuronal cell lines and synaptosomes, 29(2) Toxicon261-264 (1991); and Tei-ichi Nishiki et al., Identification of proteinreceptor for Clostridium botulinum type B neurotoxin in rat brainsynaptosomes, 269(14) J. Biol. Chem. 10498-10503 (1994). Othernon-limiting assays include immunocytochemical assays that detect toxinbinding using labeled or unlabeled antibodies, see, e.g., AtsushiNishikawa et al., The receptor and transporter for internalization ofClostridium botulinum type C progenitor toxin into HT-29 cells, 319(2)Biochem. Biophys. Res. Commun. 327-333 (2004) and immunoprecipitationassays, see, e.g., Yukako Fujinaga et al., Molecular characterization ofbinding subcomponents of Clostridium botulinum type C progenitor toxinfor intestinal epithelial cells and erythrocytes, 150(Pt 5) Microbiology1529-1538 (2004). Antibodies useful for these assays include, withoutlimitation, antibodies selected against a BoNT/A, antibodies selectedagainst a BoNT/A receptor, such as, e.g., FGFR3, antibodies selectedagainst a ganglioside, such as, e.g., GD1a, GD1b, GD3, GQ1b, or GT1b andselected against a test compound, such as, e.g., a molecule thatselectively binds a BoNT/A receptor wherein selective binding modulatesBoNT/A activity. If the antibody is labeled, the binding of the moleculecan be detected by various means, including Western blotting, directmicroscopic observation of the cellular location of the antibody,measurement of cell or substrate-bound antibody following a wash step,or electrophoresis, employing techniques well-known to those of skill inthe art. If the antibody is unlabeled, one may employ a labeledsecondary antibody for indirect detection of the bound molecule, anddetection can proceed as for a labeled antibody. It is understood thatthese and similar assays that determine BoNT/A uptake properties orcharacteristics can be useful in detecting BoNT/A activity.

Assays that monitor the release of a molecule after exposure to BoNT/Acan also be used to assess for the presence of BoNT/A activity. In theseassays, inhibition of the molecule's release would occur in cellsexpressing a FGFR3 after BoNT/A treatment. As a non-limiting example theinhibition of insulin release assay disclosed in the presentspecification can monitor the release of a molecule after exposure toBoNT/A and thereby be useful in assessing whether a molecule selectivelybinds a BoNT/A receptor (see Example I). Other non-limiting assaysinclude methods that measure inhibition of radio-labeled catecholaminerelease from neurons, such as, e.g., [³H]noradrenaline or [³H]dopaminerelease, see e.g., A Fassio et al., Evidence for calcium-dependentvesicular transmitter release insensitive to tetanus toxin and botulinumtoxin type F, 90(3) Neuroscience 893-902 (1999); and Sara Stigliani etal., The sensitivity of catecholamine release to botulinum toxin C1 andE suggests selective targeting of vesicles set into the readilyreleasable pool, 85(2) J. Neurochem. 409-421 (2003), or measurescatecholamine release using a fluorometric procedure, see, e.g., Antonde Paiva et al., A role for the interchain disulfide or itsparticipating thiols in the internalization of botulinum neurotoxin Arevealed by a toxin derivative that binds to ecto-acceptors and inhibitstransmitter release intracellularly, 268(28) J. Biol. Chem. 20838-20844(1993); Gary W. Lawrence et al., Distinct exocytotic responses of intactand permeabilised chromaffin cells after cleavage of the 25-kDasynaptosomal-associated protein (SNAP-25) or synaptobrevin by botulinumtoxin A or B, 236(3) Eur. J. Biochem. 877-886 (1996); and Patrick Foranet al., Botulinum neurotoxin C1 cleaves both syntaxin and SNAP-25 inintact and permeabilized chromaffin cells: correlation with its blockadeof catecholamine release, 35(8) Biochemistry 2630-2636 (1996); andmethods that measure inhibition of hormone release from endocrine cells,such as, e.g., anterior pituitary cells or ovarian cells. It isunderstood that these and similar assays for molecule release can beuseful in assessing BoNT/A activity.

As non-limiting examples, an inhibition of insulin release assay can beused to determine the presence of BoNT/A activity in cells containing aFGFR3 and capable of secreting insulin; an inhibition of noradrenalinerelease assay can be used to determine BoNT/A activity in cellscontaining a FGFR3 and capable of secreting noradrenaline; and aninhibition of estrogen release assay can be used to determine BoNT/Aactivity in cells containing a FGFR3 and capable of secreting estrogen.

Assays that detect the cleavage of a BoNT/A substrate after exposure toBoNT/A can also be used to assess for the presence of BoNT/A activity.In these assays, generation of a BoNT/A cleavage-product would bedetected after BoNT/A treatment. As a non-limiting example the SNAP-25cleavage assay disclosed in the present specification can detect thecleavage of a BoNT/A substrate after exposure to BoNT/A and thereby beuseful in assessing BoNT/A activity (see Example I). Other non-limitingmethods useful to detect the cleavage of a BoNT/A substrate afterexposure to BoNT/A are described in, e.g., Lance E. Steward et al., FRETProtease Assays for Botulinum Serotype A/E Toxins, U.S. PatentPublication No. 2003/0143650 (Jul. 31, 2003); and Ester Fernandez-Salaset al., Cell-based Fluorescence Resonance Energy Transfer (FRET) Assaysfor Clostridial Toxins, U.S. Patent Publication 2004/0072270 (Apr. 15,2004). It is understood that these and similar assays for BoNT/Asubstrate cleavage can be useful in assessing BoNT/A activity.

As non-limiting examples, western blot analysis using an antibody thatrecognizes BoNT/A SNAP-25-cleaved product can be used to determine thepresence of BoNT/A activity. Examples of anti-SNAP-25 antibodies usefulfor these assays include, without limitation, rabbit polyclonalanti-SNAP25₁₉₇ antiserum pAb anti-SNAP25197 #1 (Allergan, Inc., Irvine,Calif.), mouse monoclonal anti-SNAP-25 antibody SMI-81 (SternbergerMonoclonals, Lutherville, Md.), mouse monoclonal anti-SNAP-25 antibodyC171.1 (Synaptic Systems, Goettingen, Germany), mouse monoclonalanti-SNAP-25 antibody C171.2 (Synaptic Systems, Goettingen, Germany),mouse monoclonal anti-SNAP-25 antibody SP12 (Abcam, Cambridge, Mass.),rabbit polyclonal anti-SNAP-25 antiserum (Synaptic Systems, Goettingen,Germany), and rabbit polyclonal anti-SNAP-25 antiserum (Abcam,Cambridge, Mass.).

The methods disclosed in the present specification include, in part, asample. As used herein, the term “sample” means any biological matterthat contains or potentially contains an active BoNT/A. A variety ofsamples can be assayed according to a method disclosed in the presentspecification including, without limitation, purified, partiallypurified, or unpurified BoNT/A; recombinant single chain or di-chaintoxin with a naturally or non-naturally occurring sequence; recombinantBoNT/A with a modified protease specificity; recombinant BoNT/A with analtered cell specificity; chimeric toxin containing structural elementsfrom multiple BoNT/A species or subtypes; bulk BoNT/A; formulated BoNT/Aproduct; and foods; cells or crude, fractionated or partially purifiedcell lysates, for example, engineered to include a recombinant nucleicacid encoding a BoNT/A; bacterial, baculoviral and yeast lysates; raw,cooked, partially cooked or processed foods; beverages; animal feed;soil samples; water samples; pond sediments; lotions; cosmetics; andclinical formulations. It is understood that the term sample encompassestissue samples, including, without limitation, mammalian tissue samples,livestock tissue samples such as sheep, cow and pig tissue samples;primate tissue samples; and human tissue samples. Such samplesencompass, without limitation, intestinal samples such as infantintestinal samples, tissue samples obtained from a wound. Other suchsamples include mammalian tissue, mammalian saliva, mammalian excretionsand mammalian feces. As non-limiting examples, a method of the inventioncan be useful for detecting the presence or activity of a BoNT/A in afood or beverage sample; to assay a sample from a human or animal, forexample, exposed to a BoNT/A or having one or more symptoms of a BoNT/Aexposure; to follow activity during production and purification ofBoNT/A; or to assay formulated BoNT/A products such as pharmaceuticalsor cosmetics.

It is envisioned that a wide variety of processing formats can be usedin conjunction with the methods disclosed present specification,including, without limitation, manual processing, partialautomated-processing, semi-automated-processing, fullautomated-processing, high throughput processing, high contentprocessing, and the like or any combination thereof.

Other aspect of the present invention provide methods of reducing BoNT/Aactivity in a human comprising administering to said human apharmaceutical composition comprising a molecule that selectively bindsa FGFR3 wherein said selective binding reduces the ability of BoNT/A tobind to said FGFR3. In is envisioned that any molecule that canselectively bind to a FGFR3 in a manner that prevents BoNT/A binding tothat same FGFR3 can be useful, including, without limitation, ananti-FGFR3 antibody, an FGF or an FGF agonist. In addition, a FGFR3, aFGFR3 fragment retaining BoNT/A selective binding activity, orpeptidomimetic thereof can also be useful. Molecules that selectivelybinds a FGFR3, and thus useful in methods of reducing BoNT/A activityare described in, e.g., Avner Yayon et al., Antibodies that blockreceptor protein tyrosone kinase activation, methods of screening forand using thereof, International Publication No. WO 02/102972 (Dec. 27,2002); Avner Yayon et al., Antibodies that block receptor proteintyrosone kinase activation, methods of screening for and using thereof,International Publication No. WO 02/102973 (Dec. 27, 2002); andElisabeth Thomassen-Wolf et al., Antibodies that block receptor proteintyrosone kinase activation, methods of screening for and using thereof,International Publication No. WO 02/102854 (Dec. 27, 2002)

Aspects of the present invention provide, in part, a method of reducingBoNT/A activity in a human by administering a pharmaceutical compositioncomprising a molecule that selectively binds a FGFR3. The administeredcomposition can be formulated in a variety of pharmaceuticallyacceptable media, as described below. An effective dose of a compositiondisclosed in the present specification will depend upon the particularmolecule selected, the route administration, and the particularcharacteristics of the human or other mammal, such as age, weight,general health and the like. An effective dose can be determined in ananimal model prior to administration to humans. Compositions useful inaspects of the invention can be administered by a variety of routes tostimulate an immune response. As a non-limiting example, oral toleranceis well-recognized in the art (see, for example, Weiner, HospitalPractice, pp. 53-58 (Sep. 15, 1995). Those skilled in the art canreadily determine for a particular composition, a suitablepharmacological composition, an appropriate antigen payload; route ofadministration; volume of dose; and pharmaceutical regimen useful in aparticular animal, for example, humans.

As disclosed herein a pharmaceutical composition is administered to ahuman or other mammal to reduce BoNT/A activity. As used herein, theterm “reduce,” when used in reference to administering to a human orother mammal an effective amount of a pharmaceutical composition, meansreducing a symptom of a condition characterized by exposure BoNT/Aactivity, or delaying or preventing onset of a symptom of a conditioncharacterized by exposure to BoNT/A activity in the human or othermammal. For example, the term “reducing” can mean reducing a symptom ofa condition characterized by exposure to BoNT/A activity by at least30%, 40%, 60%, 70%, 80%, 90% or 100%. The effectiveness of apharmaceutical composition in treating a condition characterized byexposure to BoNT/A activity can be determined by observing one or moreclinical symptoms or physiological indicators associated with thecondition. An improvement in a condition characterized by exposure toBoNT/A activity also can be indicated by a reduced need for a concurrenttherapy. Those of skill in the art will know the appropriate symptoms orindicators associated with specific conditions and will know how todetermine if a human or other mammal is a candidate for treatment with apharmaceutical composition disclosed in the present specification. Inparticular, it is understood that those skilled in the art will be ableto determine if a condition if characterized by exposure BoNT/Aactivity, for example, by comparison of levels of BoNT/A activity fromthe human or other mammal with a normal control cells.

The appropriate effective amount to be administered for a particularapplication of the methods can be determined by those skilled in theart, using the guidance provided herein. For example, an effectiveamount can be extrapolated from assays as described herein above. Oneskilled in the art will recognize that the condition of the patient canbe monitored throughout the course of therapy and that the effectiveamount of a composition that is administered can be adjustedaccordingly.

A pharmaceutical composition useful in aspects of the inventiongenerally is administered in a pharmaceutical acceptable composition. Asused herein, the term “pharmaceutically acceptable” refer to anymolecular entity or composition that does not produce an adverse,allergic or other untoward or unwanted reaction when administered to ahuman or other mammal. As used herein, the term “pharmaceuticallyacceptable composition” refers to a therapeutically effectiveconcentration of an active ingredient. A pharmaceutical composition maybe administered to a patient alone, or in combination with othersupplementary active ingredients, agents, drugs or hormones. Thepharmaceutical compositions may be manufactured using any of a varietyof processes, including, without limitation, conventional mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping, and lyophilizing. The pharmaceuticalcomposition can take any of a variety of forms including, withoutlimitation, a sterile solution, suspension, emulsion, lyophilizate,tablet, pill, pellet, capsule, powder, syrup, elixir or any other dosageform suitable for administration.

It is also envisioned that a pharmaceutical composition disclosed in thepresent specification can optionally include a pharmaceuticallyacceptable carriers that facilitate processing of an active ingredientinto pharmaceutically acceptable compositions. As used herein, the term“pharmacologically acceptable carrier” refers to any carrier that hassubstantially no long term or permanent detrimental effect whenadministered and encompasses terms such as “pharmacologically acceptablevehicle, stabilizer, diluent, auxiliary or excipient.” Such a carriergenerally is mixed with an active compound, or permitted to dilute orenclose the active compound and can be a solid, semi-solid, or liquidagent. It is understood that the active ingredients can be soluble orcan be delivered as a suspension in the desired carrier or diluent. Anyof a variety of pharmaceutically acceptable carriers can be usedincluding, without limitation, aqueous media such as, e.g., distilled,deionized water, saline; solvents; dispersion media; coatings;antibacterial and antifungal agents; isotonic and absorption delayingagents; or any other inactive ingredient. Selection of apharmacologically acceptable carrier can depend on the mode ofadministration. Except insofar as any pharmacologically acceptablecarrier is incompatible with the active ingredient, its use inpharmaceutically acceptable compositions is contemplated. Non-limitingexamples of specific uses of such pharmaceutical carriers can be foundin PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS (Howard C.Ansel et al., eds., Lippincott Williams & Wilkins Publishers, 7th ed.1999); REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (Alfonso R.Gennaro ed., Lippincott, Williams & Wilkins, 20 ed. 2000); GOODMAN &GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS (Joel G. Hardman etal., eds., McGraw-Hill Professional, 10^(th) ed. 2001); and HANDBOOK OFPHARMACEUTICAL EXCIPIENTS (Raymond C. Rowe et al., APhA Publications, 4edition 2003). These protocols are routine procedures and anymodifications are well within the scope of one skilled in the art andfrom the teaching herein.

It is further envisioned that a pharmaceutical composition disclosed inthe present specification can optionally include, without limitation,other pharmaceutically acceptable components, including, withoutlimitation, buffers, preservatives, tonicity adjusters, salts,antioxidants, physiological substances, pharmacological substances,bulking agents, emulsifying agents, wetting agents, sweetening orflavoring agents, and the like. Various buffers and means for adjustingpH can be used to prepare a pharmaceutical composition disclosed in thepresent specification, provided that the resulting preparation ispharmaceutically acceptable. Such buffers include, without limitation,acetate buffers, citrate buffers, phosphate buffers, neutral bufferedsaline, phosphate buffered saline and borate buffers. It is understoodthat acids or bases can be used to adjust the pH of a composition asneeded. Pharmaceutically acceptable antioxidants include, withoutlimitation, sodium metabisulfite, sodium thiosulfate, acetylcysteine,butylated hydroxyanisole and butylated hydroxytoluene. Usefulpreservatives include, without limitation, benzalkonium chloride,chlorobutanol, thimerosal, phenylmercuric acetate, phenylmercuricnitrate and a stabilized oxy chloro composition, for example, PURITE®.Tonicity adjustors useful in a pharmaceutical composition include,without limitation, salts such as, e.g., sodium chloride, potassiumchloride, mannitol or glycerin and other pharmaceutically acceptabletonicity adjustor. The pharmaceutical composition may be provided as asalt and can be formed with many acids, including but not limited to,hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc.Salts tend to be more soluble in aqueous or other protonic solvents thanare the corresponding free base forms. It is understood that these andother substances known in the art of pharmacology can be included in apharmaceutical composition useful in the invention.

A pharmaceutical composition useful in a method of the disclosure isadministered to a human or other mammal in an effective amount. Such aneffective amount generally is the minimum dose necessary to achieve thedesired therapeutic effect, which can be, for example, that amountroughly necessary to reduce the symptoms associated with exposure toBoNT/A activity. For example, the term “effective amount” when used withrespect to treating exposure to BoNT/A activity can be a dose sufficientto the symptoms, for example, by at least 30%, 40%, 50%, 60%, 70%, 80%,90% or 100%. Such a dose generally is in the range of 0.1-1000 mg/dayand can be, for example, in the range of 0.1-500 mg/day, 0.5-500 mg/day,0.5-100 mg/day, 0.5-50 mg/day, 0.5-20 mg/day, 0.5-10 mg/day or 0.5-5mg/day, with the actual amount to be administered determined by aphysician taking into account the relevant circumstances including theseverity of the BoNT/A exposure, the age and weight of the patient, thepatient's general physical condition, the cause of the BoNT/A exposureand the route of administration. Where repeated administration is used,the frequency of administration depends, in part, on the half-life ofthe pharmaceutical composition. Suppositories and extended releaseformulations can be useful in the invention and include, for example,dermal patches, formulations for deposit on or under the skin andformulations for intramuscular injection. It is understood thatslow-release formulations also can be useful in the methods of theinvention. The subject receiving the pharmaceutical composition can beany mammal or other vertebrate capable of experiencing exposure toBoNT/A activity, for example, a human, primate, horse, cow, dog, cat orbird.

Various routes of administration can be useful for reducing BoNT/Aactivity according to a method of the invention. A pharmaceuticalcomposition useful in the methods of the invention can be administeredto a mammal by any of a variety of means depending, for example, on thetype and location of BoNT/A exposure to be treated, the pharmaceuticalcomposition, or other compound to be included in the composition, andthe history, risk factors and symptoms of the subject. Routes ofadministration suitable for the methods of the invention include bothsystemic and local administration. As non-limiting examples, apharmaceutical composition useful for reducing BoNT/A activity can beadministered orally or by subcutaneous pump; by dermal patch; byintravenous, subcutaneous or intramuscular injection; by topical drops,creams, gels or ointments; as an implanted or injected extended releaseformulation; as a bioerodible or non-bioerodible delivery system; bysubcutaneous minipump or other implanted device; by intrathecal pump orinjection; or by epidural injection. An exemplary list of biodegradablepolymers and methods of use are described in, e.g., HANDBOOK OFBIODEGRADABLE POLYMERS (Abraham J. Domb et al., eds., OverseasPublishers Association, 1997); CONTROLLED DRUG DELIVERY: DESIGNINGTECHNOLOGIES FOR THE FUTURE (Kinam Park & Randy J. Mrsny eds., AmericanChemical Association, 2000); Vernon G. Wong, Method for Reducing orPreventing Transplant Rejection in the Eye and Intraocular Implants forUse Therefor, U.S. Pat. No. 6,699,493 (Mar. 2, 2004); Vernon G. Wong &Mae W. L. Hu, Methods for Treating Inflammation-mediated Conditions ofthe Eye, U.S. Pat. No. 6,726,918 (Apr. 27, 2004); David A. Weber et al.,Methods and Apparatus for Delivery of Ocular Implants, U.S. PatentPublication No.

US2004/0054374 (Mar. 18, 2004); Thierry Nivaggioli et al., BiodegradableOcular Implant, U.S. Patent Publication No. US2004/0137059 (Jul. 15,2004). It is understood that the frequency and duration of dosing willbe dependent, in part, on the relief desired and the half-life of thetolerogizing composition.

In particular embodiments, a method of the invention is practiced byperipheral administration of a pharmaceutical composition. As usedherein, the term “peripheral administration” or “administeredperipherally” means introducing an agent into a subject outside of thecentral nervous system. Peripheral administration encompasses any routeof administration other than direct administration to the spine orbrain. As such, it is clear that intrathecal and epidural administrationas well as cranial injection or implantation are not within the scope ofthe term “peripheral administration” or “administered peripherally.”

Peripheral administration can be local or systemic. Local administrationresults in significantly more of a pharmaceutical composition beingdelivered to and about the site of local administration than to regionsdistal to the site of administration. Systemic administration results indelivery of a pharmaceutical composition to essentially the entireperipheral nervous system of the subject and may also result in deliveryto the central nervous system depending on the properties of thecomposition.

Routes of peripheral administration useful in the methods of theinvention encompass, without limitation, oral administration, topicaladministration, intravenous or other injection, and implanted minipumpsor other extended release devices or formulations. A pharmaceuticalcomposition useful in the invention can be peripherally administered,for example, orally in any acceptable form such as in a tablet, liquid,capsule, powder, or the like; by intravenous, intraperitoneal,intramuscular, subcutaneous or parenteral injection; by transdermaldiffusion or electrophoresis; topically in any acceptable form such asin drops, creams, gels or ointments; and by minipump or other implantedextended release device or formulation.

Other aspect of the present invention provide methods of screening for amolecule able to compete with BoNT/A for selective binding to cellssusceptible to BoNT/A intoxication by contacting said sample with acomposition comprising an FGFR3 and detecting whether said moleculeselectively binds said FGFR3, wherein selective binding of said moleculeto said FGFR3 indicates that said molecule is able to compete withBoNT/A for selective binding to cells susceptible to BoNT/Aintoxication, and wherein if said molecule is BoNT/A, said method doesnot comprise an LD₅₀ assay. As used herein, the term “selective” bindingmeans that a binding agent is able to bind its target underphysiological conditions, or in vitro conditions substantiallyapproximating physiological conditions, to a statistically significantlygreater degree (i.e., has a smaller K_(d) or dissociation constant) thanto other, non-target molecules on the surface of the neural cell.“K_(d)” is the molar concentration of the binding agent at which halfthe target molecules are bound by the binding agent. As used herein, theterm “LD₅₀ assay” means an live animal-based in vivo assay of neurotoxinactivity comprising detecting the dose of neurotoxin at which 50% oftreated animals die, see, e.g., the Mouse Protection Assay (MPA),Charles L. Hatheway & Carol Dang, Immunogenicity of the Neurotoxins ofClostridium botulinum, 93-107 (Neurological Disease and Therapy—THERAPYWITH BOTULINUM TOXIN, Joseph Jankovic & Mark Hallett eds., MarcelDekker, 1994).

It is envisioned that any and all assay conditions suitable forscreening for a molecule able to compete with BoNT/A for selectivebinding to cells susceptible to BoNT/A intoxication can be useful,including, e.g., in vitro and in vivo assays. In addition, it is alsoforeseen that a wide variety of processing formats can be used inconjunction with the methods disclosed present specification, including,without limitation, manual processing, partial automated-processing,semi-automated-processing, full automated-processing, high throughputprocessing, high content processing, and the like or any combinationthereof.

As disclosed above, any of the methods useful for detecting BoNT/Aactivity disclosed in the present specification and any of thecompositions useful for practicing the methods useful for detectingBoNT/A activity disclosed in the present specification can be can beuseful in screening for a molecule that competes with BoNT/A for theselectively binding to a FGFR3. Thus, in aspect of this embodiment, aFGFR3 can be encoded by the nucleic acid molecule from a mammalianFGFR3, such as, e.g., a human FGFR3, a bovine FGFR3, a rat FGFR3 or amouse FGFR3; a bird FGFR3, such as, e.g., chicken FGFR3; an amphibianFGFR3, such as, e.g., a newt FGFR3 or a frog FGFR3; and a fish FGFR3,such as, e.g., a zebrafish FGFR3. In another aspect of this embodiment,a FGFR3 can be a mammalian FGFR3, such as, e.g., a human FGFR3, a bovineFGFR3, a rat FGFR3 or a mouse FGFR3; a bird FGFR3, such as, e.g.,chicken FGFR3; an amphibian FGFR3, such as, e.g., a newt FGFR3 or a frogFGFR3; and a fish FGFR3, such as, e.g., a zebrafish FGFR3. In anotheraspect of this embodiment, a FGFR3 useful in screening for a moleculethat competes with BoNT/A for the selectively binding to the FGFR3 canbe transiently or stably contained in a cell. In another aspect of thisembodiment, a composition useful in screening for a molecule thatcompetes with BoNT/A for the selectively binding to a FGFR3 comprises aFGFR3 and optionally a G1b polysialoganglioside, such as, e.g., GD1a,GD1b, GD3, GQ1b, or GT1b.

In another aspect of this embodiment, a cell can include cells, such as,e.g., neuronal cells including, without limitation, primary neuronalcells; immortalized or established neuronal cells; transformed neuronalcells; neuronal tumor cells; stably and transiently transfected neuronalcells expressing a FGFR3, and further include, yet are not limited to,mammalian, murine, rat, primate and human neuronal cells. Other aspectsof this embodiment include cells from, such as, e.g., neuronal celllines including, without limitation, neuroblastoma cell lines, neuronalhybrid cell lines, spinal cord cell lines, central nervous system celllines, cerebral cortex cell lines, dorsal root ganglion cell lines,hippocampal cell lines and pheochromocytoma cell lines. Non-limitingexamples of neuronal cell lines include, e.g., neuroblastoma cell linesBE(2)-C, BE(2)-M17, C1300, CHP-212, CHP-126, IMR 32, KELLY, LA-N-2,MC-IXC, MHH-NB-11, N18Tg2, N1E-115, N4TG3, Neuro-2A, NB41A3, NS20Y,SH-SY5Y, SIMA, SK-N-DZ, SK-N-F1, SK-N-MC and SK-N-SH;neuroblastoma/glioma hybrid cell lines N18, NG108-15 and NG115-401L;neuroblastoma/motor neuron hybrid cell lines NSC-19 and NSC-32;neuroblastoma/root ganglion neuron hybrid cell lines F11, ND-E, ND-U1,ND7/23, ND8/34 and ND27; the neuroblastoma/hippocampal neuron hybridcell line HN-33; spinal cord cell lines TE 189.T and M4b; cerebralcortex cell lines CNh, HCN-1a and HCN-2; dorsal root ganglia cell lineG4b; hippocampal cell lines HT-4, HT-22 and HN33; FGFR3 expressing celllines H929, JIM-3, KMS-11, KMS-18, LB278, LB375, LB1017, LB2100, LP-1,OPM-2, PCL1 and UTMC-2. In further aspects of this embodiment, an FGFR3expressing cell can be, e.g., H929, JIM-3, KMS-11, KMS-18, LB278, LB375,LB1017, LB2100, LP-1, OPM-2, PCL1 UTMC-2, B9, TC, L6 and CFK2. Otheraspects of this embodiment include cells, such as, e.g., non-neuronalcells including, without limitation, primary non-neuronal cells;immortalized or established non-neuronal cells; transformed non-neuronalcells; non-neuronal tumor cells; stably and transiently transfectednon-neuronal cells expressing a FGFR3, and further include, yet are notlimited to, mammalian, murine, rat, primate and human non-neuronalcells. Other aspects of this embodiment include cells, such as, e.g.,non-neuronal cells useful in aspects of the invention further include,without limitation, anterior pituitary cells; adrenal cells, pancreaticcells, ovarian cells, kidney cells, stomach cell, blood cells,epithelial cells, fibroblasts, thyroid cells, chondrocytes, musclecells, hepatocytes, glandular cells and cells involved in glucosetransporter (GLUT4) translocation.

The molecule to be tested in the screening method may be a “small”organic compound of synthetic origin, or may be a macromolecule (eitherof synthetic or biological origin) including without limitation, apolypeptide, such as, e.g., a growth factor, a neurotoxin, a modifiedneurotoxin, an antibody or an antibody derivative; a nucleic acid, suchas, e.g., a nucleic acid aptomer; and a polysaccharide, such as, e.g., aganglioside or a lectin. In one embodiment, the molecule is a syntheticmolecule designed based on the tertiary structure and three dimensionalconformation of FGF or an antibody that inhibits BoNT/A binding to aFGFR3. Such SAR (structure/activity relationship) analysis is routine inthe art of medicinal chemistry, among other fields.

A wide variety of assays can be used to determine whether a moleculeselectively binds a FGFR3, including direct and indirect assays fortoxin uptake. Assays that determine BoNT/A binding or uptake propertiescan be used to assess whether a molecule selectively binds a FGFR3. Suchassays include, without limitation, cross-linking assays using labeledBoNT/A, such as, e.g., BoNT/A-SBED, see, e.g., Example II of the presentspecification and [¹²⁵I] BoNT/A, see, e.g., Noriko Yokosawa et al.,Binding of Clostridium botulinum type C neurotoxin to differentneuroblastoma cell lines, 57(1) Infect. Immun. 272-277 (1989); NorikoYokosawa et al., Binding of botulinum type C1, D and E neurotoxins toneuronal cell lines and synaptosomes, 29(2) Toxicon 261-264 (1991); andTei-ichi Nishiki et al., Identification of protein receptor forClostridium botulinum type B neurotoxin in rat brain synaptosomes,269(14) J. Biol. Chem. 10498-10503 (1994). Other non-limiting assaysinclude immunocytochemical assays that detect toxin binding usinglabeled or unlabeled antibodies, see, e.g., Atsushi Nishikawa et al.,The receptor and transporter for internalization of Clostridiumbotulinum type C progenitor toxin into HT-29 cells, 319(2) Biochem.Biophys. Res. Commun. 327-333 (2004) and immunoprecipitation assays,see, e.g., Yukako Fujinaga et al., Molecular characterization of bindingsubcomponents of Clostridium botulinum type C progenitor toxin forintestinal epithelial cells and erythrocytes, 150(Pt 5) Microbiology1529-1538 (2004). Antibodies useful for these assays include, withoutlimitation, antibodies selected against a BoNT/A, antibodies selectedagainst a BoNT/A receptor, such as, e.g., FGFR3, antibodies selectedagainst a ganglioside, such as, e.g., GD1a, GD1b, GD3, GQ1b, or GT1b andselected against a test compound, such as, e.g., a molecule thatselectively binds a BoNT/A receptor wherein selective binding modulatesBoNT/A activity. If the antibody is labeled, the binding of the moleculecan be detected by various means, including Western blotting, directmicroscopic observation of the cellular location of the antibody,measurement of cell or substrate-bound antibody following a wash step,or electrophoresis, employing techniques well-known to those of skill inthe art. If the antibody is unlabeled, one may employ a labeledsecondary antibody for indirect detection of the bound molecule, anddetection can proceed as for a labeled antibody. It is understood thatthese and similar assays that determine BoNT/A uptake properties orcharacteristics can be useful in selecting a neuron or other cellsuseful in aspects of the invention.

Assays that monitor the release of a molecule after exposure to BoNT/Acan also be used to assess whether a molecule selectively binds a FGFR3.In these assays, inhibition of the molecule's release would occur incells expressing a FGFR3 after BoNT/A treatment. As a non-limitingexample the inhibition of insulin release assay disclosed in the presentspecification can monitor the release of a molecule after exposure toBoNT/A and thereby be useful in assessing whether a molecule selectivelybinds a FGFR3 (see Example I). Other non-limiting assays include methodsthat measure inhibition of radio-labeled catecholamine release fromneurons, such as, e.g., [³H]noradrenaline or [³H]dopamine release, seee.g., A Fassio et al., Evidence for calcium-dependent vesiculartransmitter release insensitive to tetanus toxin and botulinum toxintype F, 90(3) Neuroscience 893-902 (1999); and Sara Stigliani et al.,The sensitivity of catecholamine release to botulinum toxin C1 and Esuggests selective targeting of vesicles set into the readily releasablepool, 85(2) J. Neurochem. 409-421 (2003), or measures catecholaminerelease using a fluorometric procedure, see, e.g., Anton de Paiva etal., A role for the interchain disulfide or its participating thiols inthe internalization of botulinum neurotoxin A revealed by a toxinderivative that binds to ecto-acceptors and inhibits transmitter releaseintracellularly, 268(28) J. Biol. Chem. 20838-20844 (1993); Gary W.Lawrence et al., Distinct exocytotic responses of intact andpermeabilised chromaffin cells after cleavage of the 25-kDasynaptosomal-associated protein (SNAP-25) or synaptobrevin by botulinumtoxin A or B, 236(3) Eur. J. Biochem. 877-886 (1996); and Patrick Foranet al., Botulinum neurotoxin C1 cleaves both syntaxin and SNAP-25 inintact and permeabilized chromaffin cells: correlation with its blockadeof catecholamine release, 35(8) Biochemistry 2630-2636 (1996); andmethods that measure inhibition of hormone release from endocrine cells,such as, e.g., anterior pituitary cells or ovarian cells. It isunderstood that these and similar assays for molecule release can beuseful in assessing whether a molecule selectively binds a FGFR3.

As non-limiting examples, an inhibition of insulin release assay can beused to test whether a molecule selectively binds a FGFR3 in a FGFR3containing cells capable of secreting insulin; an inhibition ofnoradrenaline release assay using can be used to test whether a moleculeselectively binds a FGFR3 in a FGFR3 containing cells capable ofsecreting noradrenaline; and an inhibition of estrogen release assay canbe used to assay whether a molecule selectively binds a FGFR3 in a FGFR3containing cells and capable of secreting estrogen.

Assays that detect the cleavage of a BoNT/A substrate after exposure toBoNT/A can also be used to assess whether a molecule selectively binds aFGFR3. In these assays, generation of a BoNT/A cleavage-product would bedetected in cells expressing a FGFR3 after BoNT/A treatment. As anon-limiting example the SNAP-25 cleavage assay disclosed in the presentspecification can detect the cleavage of a BoNT/A substrate afterexposure to BoNT/A and thereby be useful in assessing whether a moleculeselectively binds a BoNT/A receptor (see Example I). Other non-limitingmethods useful to detect the cleavage of a BoNT/A substrate afterexposure to BoNT/A are described in, e.g., Lance E. Steward et al., FRETProtease Assays for Botulinum Serotype A/E Toxins, U.S. PatentPublication No. 2003/0143650 (Jul. 31, 2003); and Ester Fernandez-Salaset al., Cell-based Fluorescence Resonance Energy Transfer (FRET) Assaysfor Clostridial Toxins, U.S. Patent Publication 2004/0072270 (Apr. 15,2004). It is understood that these and similar assays for BoNT/Asubstrate cleavage can be useful in assessing whether a moleculeselectively binds a FGFR3.

As non-limiting examples, western blot analysis using an antibody thatrecognizes BoNT/A SNAP-25-cleaved product can be used to assay whether amolecule selectively binds a FGFR3. Examples of anti-SNAP-25 antibodiesuseful for these assays include, without limitation, rabbit polyclonalanti-SNAP25₁₉₇ antiserum pAb anti-SNAP25197 #1 (Allergan, Inc., Irvine,Calif.), mouse monoclonal anti-SNAP-25 antibody SMI-81 (SternbergerMonoclonals, Lutherville, Md.), mouse monoclonal anti-SNAP-25 antibodyCI 71.1 (Synaptic Systems, Goettingen, Germany), mouse monoclonalanti-SNAP-25 antibody CI 71.2 (Synaptic Systems, Goettingen, Germany),mouse monoclonal anti-SNAP-25 antibody SP12 (Abcam, Cambridge, Mass.),rabbit polyclonal anti-SNAP-25 antiserum (Synaptic Systems, Goettingen,Germany), and rabbit polyclonal anti-SNAP-25 antiserum (Abcam,Cambridge, Mass.).

Assays that detect competitive binding of a molecule with BoNT/A forselective binding to a FGFR3 can also be used to assess whether amolecule selectively binds a FGFR3. In these assays, a reduction inBoNT/A activity would be detected as the amount of a molecule thatcompetes with BoNT/A for selective binding to a BoNT/A would increase.In a non-limiting example, the competitive inhibition assay using FGFligands disclosed in the present specification can be used to detect thecompetitive binding of a molecule with BoNT/A for selective binding to aFGFR3 and thereby be useful in assessing whether a molecule selectivelybinds a BoNT/A receptor (see Example II). Thus in one aspect of thisembodiment, competitive binding assays using a FGFR3-binding moleculewith BoNT/A for selective binding to a FGFR3 can be used to assesswhether a molecule selectively binds a FGFR3.

Other aspect of the present invention provide methods of rendering acell susceptible to cleavage of SNARE proteins by BoNT/A, comprisinginducing said cell to express a FGFR3. Other aspect of the presentinvention provide methods of transiently rendering a cell susceptible tocleavage of SNARE proteins by BoNT/A, comprising transiently inducingsaid cell to express a FGFR3. Other aspect of the present inventionprovide methods of stably rendering a cell susceptible to cleavage ofSNARE proteins by BoNT/A, comprising stably inducing said cell toexpress a FGFR3.

Other aspect of the present invention provide methods of marketing aneurotoxin capable of selectively binding to the same FGFR3 as BoNT/Acomprising obtaining marketing approval from a governmental or regionalregulatory authority for a therapeutic neurotoxin, wherein saidneurotoxin is assayed for selective binding to a cell comprisingcontacting said neurotoxin with a composition comprising a FGFR3 anddetecting whether said neurotoxin selectively binds said FGFR3, whereinselective binding of said neurotoxin to said FGFR3 indicates that saidneurotoxin is able to selective binding to cells susceptible to BoNT/Aintoxication and wherein if said molecule is BoNT/A, said method doesnot comprise an LD₅₀ assay; packaging said neurotoxin for sale in amanner consistent with the requirements of said regulatory authority,and selling said neurotoxin.

Other aspect of the present invention provide methods of marketing aneurotoxin capable of selectively binding to the same FGFR3 as BoNT/Acomprising obtaining marketing approval from a governmental or regionalregulatory authority for a therapeutic neurotoxin, wherein saidneurotoxin is assayed for selective binding to a cell comprisingcontacting said neurotoxin to a cell that contains an exogenous FGFR3wherein said contacted cell is capable of BoNT/A intoxication anddetecting the presence of BoNT/A activity of said contacted cellrelative to a control cell, where a difference in said BoNT/A activityof said contacted cell as compared to said control cell is indicative ofBoNT/A activity; packaging said neurotoxin for sale in a mannerconsistent with the requirements of said regulatory authority, andselling said neurotoxin.

In another embodiment, the invention is drawn to a polypeptidecomprising at least the H_(C) region of BONT/A, which is produced from abulk or formulated preparation wherein the bulk or formulatedpreparation is assayed for specific binding to neural cells using amethod comprising contacting said polypeptide with a compositioncomprising FGFR3 receptor and, optionally, GT1b ganglioside, anddetecting whether said polypeptide selectively binds FGFR3.

In another embodiment similar to the above aspect of the invention, thepolypeptide comprises at least an FGFR3 binding domain, other than theH_(C) domain of BoNT/A. Such a binding domain may comprise, for example,an FGF, such as FGF 1, FGF2, FGF4, FGF8 or FGF 9, or an anti-FGFR3antibody. Further, the polypeptide may optionally contain atranslocation domain such as the H_(N) domain of BoNT/A. Additionally,the polypeptide will generally contain a clostridial neurotoxin lightchain or variation thereof—the nature and/or source of the light chaincan provide differences in the extent and half-life of the therapeuticeffect of the polypeptide.

Thus, in this embodiment the claimed polypeptide is produced (whichproduction may include purification, enzymatic treatment, and/oroxidation steps) from a bulk or formulation preparation. In oneembodiment the preparation may be, for example, a cell lysate fromfermentation of a BoNT/A-producing strain of Clostridium botulinum, orfrom a suitable mammalian, insect or bacterial host cell producing arecombinant version of BoNT/A. Such a bulk preparation may also beproduced using cell-free transcription methodologies. In anotherembodiment the preparation may be purified BoNT/A formulated withassociated stabilizing proteins, such as serum albumin. In each case,the preparation may comprise BoNT/A molecules which are denatured orotherwise incorrectly folded so as not to bind to the target cells. Thepotency and/or specific activity of the preparation, or of fractionspurified from the preparation, can be detected by using the claimedassay method.

Alternatively, the polypeptide to be assayed may comprise only a portionof the entire BoNT/A molecule. For example, the bulk preparation maycontain only the heavy chain of BoNT/A, as separate production of theheavy and light chains of the toxin may be a preferred way of avoidingaccidental exposure to the neurotoxin by laboratory workers.

As another example of the above embodiment, the polypeptide may comprisea chimeric recombinant polypeptide which contains the H_(C) region ofthe heavy chain of BoNT/A (or some other FGFR3-binding moiety, such asFGF itself). The chimeric polypeptide comprises amino acid sequenceregions additional to, or other than, those present in the wild-typeBoNT/A BoNT/A molecule. For example, botulinum and tetanus toxins may beused as the basis for the creation of transport proteins, see, e.g.,James Oliver Dolly et al., Modification of clostridial toxins for use astransport proteins, U.S. Pat. No. 6,203,794 (Mar. 20, 2001). The lightchain of these transport proteins are generally either replaced by atherapeutic moiety or inactivated and coupled to such a therapeuticmoiety. Additionally, chimeric neurotoxins can be made comprisingpolypeptides containing domains of more than one neurotoxin see, e.g.,James Oliver Dolly et al., Activatable Recombinant Neurotoxins,International Publication No. WO 01/14570 (Mar. 1, 2001). Thus, thisaspect of the invention also encompasses, as a embodiment, chimericneurotoxins containing at least the H_(C) domain of BoNT/A. Suchmolecules may be useful in modulating the time or extent of theinhibition of secretory vesicle release. Further, it may be desirable totarget agents, such as therapeutic agents, to the extracellular surfaceof the neural cell membrane. Thus, such an agent may be joined (e.g., asa fusion protein or via post translational conjugation) to the H_(C)portion of BoNT/A. In such a case the cell lysate or conjugationreaction mixture may comprise a batch preparation in accordance withthis aspect of the invention.

The above-referenced polypeptides are screened for binding and/orinternalization essentially as mentioned above in the describedscreening method embodiment.

In yet another embodiment, the present invention is drawn to a method ofmarketing a polypeptide which contains a region capable of binding theFGFR3 receptor comprising obtaining permission from a governmental orregional drug regulatory authority to sell said polypeptide, whereinsaid polypeptide is first produced from a bulk preparation which isassayed for selective binding of said polypeptide to neural cells bycontacting the bulk preparation containing said polypeptide with acomposition comprising FGFR3 receptor, and optionally GT1b ganglioside,and detecting whether said polypeptide selectively binds FGFR3 undersuch conditions, packaging said polypeptide for sale in a mannerconsistent with the requirements of said regulatory authority, andoffering said polypeptide for sale.

In this embodiment the invention is drawn to a method of marketing apolypeptide containing the H_(C) region of a BoNT/A toxin. Thepolypeptide at issue in this embodiment of the invention is producedfrom a bulk preparation which is assayed for purity or activity usingthe screening method described previously. In a step of this method,permission is obtained from a regulatory body for the marketing of suchpolypeptide. In this context “permission” may be tacit or express; thatis, permission or approval may be obtained from the regulatory authorityfor the sale of a therapeutic agent or composition comprising saidpolypeptide, in which case “permission” is marketing approval for thesale of such agent or composition. Alternatively, “permission”, as usedherein, may comprise the assent, either affirmatively given ormanifested by its lack of objection, of such regulatory authority to thecontinued sale of a product containing a polypeptide assayed in this newmanner. As before, the polypeptide may comprise BoNT/A, or a derivativethereof, or a fusion protein or conjugate containing the H_(C) region ofthe BoNT/A heavy chain.

The therapeutic product comprising the polypeptide originally containedin the bulk preparation so assayed is labeled in accordance with therequirements of the regulatory authority. The product is then offeredfor sale. Offering for sale may comprise advertising or sales activity,educational seminars directed at doctors, hospitals, insurers, orpatients, conversations with state, regional or governmental officialsconcerning subsidy reimbursement (such as Medicare or Medical).

EXAMPLES Example I Identification of a BoNT/A Receptor Using a GeneticComplementation Procedure

1. Identification of Cells Useful in Screening for a BoNT/A Receptor

1a. Identification of BoNT/A Receptor Lacking Cells Using an InhibitionAssay for Insulin Release

To determine whether HIT-T15 cells express a receptor for BoNT/A, aninhibition assay for insulin release was performed. In response toglucose stimulation, the hamster insulinoma cell line HIT-T15 secretesinsulin in a exocytic process that depends on the activity of SNAP-25for vesicle docking and fusion. If HIT-T15 cells lack a BoNT/A receptor,these cells would be unable to uptake BoNT/A upon exposure to this toxinand insulin secretion could occur in the presence of high glucose in themedia. However, if HIT-T15 cells contain a BoNT/A receptor, insulinsecretion would be inhibited after BoNT/A treatment since the toxincould intoxicate the cell and cleave SNAP-25.

To conduct an inhibition assay for insulin release, a suitable seeddensity of approximately 1.5×10⁵ cells/mL of HIT-T15 cells was platedinto individual wells of 6-well, poly-D-lysine/Laminin coated, tissueculture plates containing 3 mL of complete Dulbecco's Modified EagleMedia (DMEM), supplemented with 10% fetal bovine serum (FBS), 1×penicillin/streptomycin solution (Invitrogen, Inc, Carlsbad, Calif.) and4 mM Glutamine (Invitrogen, Inc, Carlsbad, Calif.), and grown in a 37°C. incubator under 5% carbon dioxide until the cells reach a density ofabout 5×10⁵ cells/ml (6-16 hours). A group of HIT-T15 cells were treatedwith approximately 1 nM of PURE-A by introducing the toxin usingelectroporation using a GENE PULSER® II set at 960 μF and 0.28 kV(Bio-Rad Laboratories, Hercules, Calif.). An untreated control groupunderwent electroporation without PURE-A. The media from the wellscontaining treated and untreated electroporated cells was replaced with3 mL of fresh complete DMEM supplement with either 5.6 mM glucose (lowglucose) or 25 mM glucose (high glucose) and these cells were incubatedin a 37° C. incubator under 5% carbon dioxide for approximately 1 hourto induce insulin secretion. The conditioned media was transferred to 15mL tubes and the amount of insulin present in the condition mediasamples was determined using an Insulin ELISA assay (PeninsulaLaboratories, Inc., San Carlos, Calif.). Exocytosis is expressed as theamount of insulin secreted per 1.5×10⁵ cell/hr. Insulin release wasdetected in BoNT/A-untreated cells simulated by 25 mM glucose, butinsulin secretion was inhibited in BoNT/A-treated cells (see FIG. 3 a).These data indicate that the release of insulin in HIT-T15 cells ismediated, in part, by SNAP-25, but that these cells lack a BoNT/Areceptor.

1b. Identification of BoNT/A Receptor Lacking Cells Using an Using aSNAP-25 Cleavage Assay

To determine whether HIT-T15 cells express a receptor for BoNT/A, aSNAP-25 cleavage assay was performed. If HIT-T15 cells lack a BoNT/Areceptor, then only the presence of the uncleaved SNAP-25 substratewould be detected after Western blot analysis. However, if HIT-T15 cellscontain a BoNT/A receptor, then the toxin could intoxicate the cell andthe presence of the cleaved BoNT/A SNAP-25₁₉₇ product would be detected.

To conduct a SNAP-25 cleavage assay, cells were grown inpoly-D-lysine/Laminin coated 6-well plates and treated with PURE-A asdescribed above in Example I, 1a. Cells were collected in 15 ml tubes,washed once with 1 ml of phosphate-buffered saline, pH 7.4, and thentransferred to 1.5 ml microcentrifuge tubes. Cells were lysed in 0.5 mlof lysis buffer containing 50 mM N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), pH 6.8, 150 mM sodiumchloride, 1.5 mM magnesium chloride, 1 mM ethylene glycolbis(β-aminoethyl ether)N,N,N′,N′-tetraacetic acid (EGTA), 10% glyceroland 1% (v/v) Triton-X® 100 (4-octylphenol polyethoxylate), with rotationfor 1 hour at 4° C. Lysed cells were centrifuged at 5000 rpm for 10 minat 4° C. to eliminate debris and the supernatants were transferred tofresh siliconized tubes. Protein concentrations were measured byBradford's method and resuspended in 1×SDS sample buffer at 1 mg/mL orhigher concentration.

To detect for the presence of a cleaved BoNT/A substrate, samples wereboiled for 5 min, and 40 μl aliquots were separated by MOPSpolyacrylamide gel electrophoresis using NuPAGE® Novex 4-12% Bis-Trisprecast polyacrylamide gels (Invitrogen, Inc, Carlsbad, Calif.) underdenaturing, reducing conditions. Separated peptides were transferredfrom the gel onto polyvinylidene fluoride (PVDF) membranes (Invitrogen,Inc, Carlsbad, Calif.) by Western blotting using a Trans-Blot® SDsemi-dry electrophoretic transfer cell apparatus (Bio-Rad Laboratories,Hercules, Calif.). PVDF membranes were blocked by incubating at roomtemperature for 2 hours in a solution containing 25 mM Tris-BufferedSaline (25 mM 2-amino-2-hydroxymethyl-1,3-propanediol hydrochloric acid(Tris-HCl) (pH 7.4), 137 mM sodium chloride, 2.7 mM potassium chloride),0.1% TWEEN-200, polyoxyethylene (20) sorbitan monolaureate, 2% bovineserum albumin, 5% nonfat dry milk. Blocked membranes were incubated at4° C. for overnight in Tris-Buffered Saline TWEEN-200 (25 mMTris-Buffered Saline, 0.1% TWEEN-20®, polyoxyethylene (20) sorbitanmonolaureate) containing a 1:5,000 dilution of rabbit polyclonalanti-SNAP-25 antiserum pAb anti-SNAP25197 #1, a polyclonal antibodywhich is specific for the SNAP25₁₉₇-cleavage product and does notcross-react with full-length SNAP25₂₀₆, (Allergan, Inc., generated undercontract with Zymed Laboratories Inc., South San Francisco, Calif.).Primary antibody probed blots were washed three times for 15 minuteseach time in Tris-Buffered Saline TWEEN-20®. Washed membranes wereincubated at room temperature for 2 hours in Tris-Buffered SalineTWEEN-20® containing a 1:20,000 dilution of goat polyclonal anti-rabbitimmunoglobulin G, heavy and light chains (IgG, H+L) antibody conjugatedto horseradish peroxidase (HRP; Pierce Biotechnology, Inc., Rockford,Ill.) as a secondary antibody. Secondary antibody-probed blots werewashed three times for 15 minutes each time in Tris-Buffered SalineTWEEN-20®. Signal detection of the labeled BoNT/A SNAP25₁₉₇-cleavageproduct was visualized using the ECL Plus™ Western Blot Detection System(Amersham Biosciences, Piscataway, N.J.) and the membrane was imaged andcleavage product quantitated with a Typhoon 9410 Variable Mode Imagerand Imager Analysis software (Amersham Biosciences, Piscataway, N.J.).The choice of pixel size (100 to 200 pixels) and PMT voltage settings(350 to 600, normally 400) depended on the individual blot. A BoNT/ASNAP25₁₉₇-cleavage product was detected in HIT-T15 cell treated withBoNT/A but not untreated cells, indicating that HIT-T15 cells expressSNAP-25 but not the BoNT/A receptor (see FIG. 3 b).

1c. Assessment of BoNT/A Exposure on HIT-T15 Growth

To evaluate if the presence of the toxin in the cells affect cellgrowth, HIT-T15 cells were electroporated as described above in ExampleI, 1a and monitored for 10 days. FIG. 4 a demonstrates that the presenceof the toxin delayed growth when compared to controls, but toxin-treatedcells were able to replicate normally after a recovery period. Cellaliquots for days 3, 5, 7 and 10 were also tested for the presence ofthe BoNT/A SNAP-25₁₉₇ cleavage product using the SNAP-25 cleavage assayas described above in Example I, 1b. FIG. 4 b shows that cleavage ofSNAP-25 was detected by Western blot analysis at all time points assayedwhen PURE-A was introduced into the cells.

2. Identification of BoNT/A Receptor Using Genetic Complementation

To identify a BoNT/A receptor, a nucleic acid molecule encoding a BoNT/Areceptor was cloned by genetic complementation. This procedure involvesintroducing a nucleic acid molecule encoding the BoNT/A receptor into acell line that does not contain the receptor naturally by retroviraltransduction, see, e.g., Mitchell H. Finer et al., Methods forProduction of High Titer Virus and High Efficiency Retroviral MediatedTransduction of Mammalian Cells, U.S. Pat. No. 5,858,740 (Jul. 12,1999).

2a. Production of a Retroviral Stock Containing pLIB ExpressionConstructs

To produce an retroviral stock containing expression constructs encodinghuman brain nucleic acid molecules, about 5×10⁵ HEK 293-based cells(AmphoPack™ 293 cells; BD Biosciences Clontech, Palo Alto, Calif.) wereplated in 60 mm tissue culture dishes containing 5 mL of completeDulbecco's Modified Eagle Media (DMEM), supplemented with 10% fetalbovine serum (FBS), 1× penicillin/streptomycin solution (Invitrogen,Inc, Carlsbad, Calif.) and 4 mM Glutamine (Invitrogen, Inc, Carlsbad,Calif.), and grown in a 37° C. incubator under 5% carbon dioxide untilthe cells reach 60% to 80% confluency or a density of about 1 to 2×10⁶cells/ml (12-24 hours). On the day of transfection, the complete,supplemented DMEM media was replaced with 3 mL of OPTI-MEM Reduced SerumMedium. A 500 μL transfection solution is prepared by adding 250 μL ofOPTI-MEM Reduced Serum Medium containing 15 μL of LipofectAmine 2000(Invitrogen, Carlsbad, Calif.) incubated at room temperature for 5minutes to 250 μL of OPTI-MEM Reduced Serum Medium containing 5 μg ofpLIB retroviral expression constructs containing nucleic acid moleculesderived from human brain cells (BD Biosciences Clontech, Palo Alto,Calif.). This transfection is incubated at room temperature forapproximately 20 minutes. The 500 μL transfection solution was thenadded to the AmphoPack™ 293 cells and the cells were incubated in a 37°C. incubator under 5% carbon dioxide for approximately 8-10 hours. Thetransfection media was replaced with 3 mL of fresh complete,supplemented DMEM and cells were incubated in a 37° C. incubator under5% carbon dioxide for approximately 48-72 hours. Theretrovirus-containing cells are harvested by detaching the cells usingthe culture media and scraping cells from the culture plate. Detachedcells and media are transferred to a 15 mL tube and centrifuged (5,000×gat 20° C. for 15 minutes) to pellet the cellular debris. The clarifiedsupernatant containing the retroviral particles is transferred to 2 mLcryovials in 1 mL aliquots and should contain approximately 5×10⁴ to5×10⁶ tu/mL of retroviral particles. Aliquots can be stored at −80° C.until needed.

2b. Transduction of Cells with a Retroviral Stock Containing pLIBExpression Constructs

To transduce cells with a retroviral stock containing expressionconstructs encoding human brain nucleic acid molecules, about 1.5×10⁵HIT-T15 cells were plated in 60 mm tissue culture dishes containing 5 mLof complete Dulbecco's Modified Eagle Media (DMEM), supplemented with10% fetal bovine serum (FBS), 1× penicillin/streptomycin solution(Invitrogen, Inc, Carlsbad, Calif.) and 4 mM Glutamine (Invitrogen, Inc,Carlsbad, Calif.), and grown in a 37° C. incubator under 5% carbondioxide until the cells reach 60% to 80% confluency or a density ofabout 5×10⁵ cells/mL (6-16 hours). Cells are inoculated with theretroviral stock containing nucleic acid molecules derived from humanbrain cells (see Example I, 2a), using a suitable multiplicity ofinfection. Approximately 4-8 μg/mL of polybrene was then added and thecells were incubated for approximately 16-24 hours in a 37° C. incubatorunder 5% carbon dioxide. The transduction media is replaced with 5 mL offresh complete, supplemented DMEM and the cells were incubated in a 37°C. incubator under 5% carbon dioxide for approximately four days. Thetransduced cells were then used to conduct a screening assay to identifya BoNT/A receptor. For greater details on procedures described in thisexample, see Retroviral Gene Transfer and Expression User ManualPT3132-1 (PR43789), BD Biosciences Clontech, Palo Alto, Calif., (Mar. 3,2004).

2c. Screening of HIT-T15 Cells Expressing a Retroviral cDNA Library

To screen for cells expressing a BoNT/A receptor, transduced HIT-T15cells as described above in Example I, 2b were screened based on theirability to bind Dynex Beads coated with Pure A (ref). Approximately 7.5mg of Dynabeads® magnetic beads (Dynal Biotechnology, LLC, Brown Deer,Wis.) coated with an antibody against the light chain of BONT/A wasadded to the media for 30 minutes at 4° C. and cells binding to theBoNT/A light chain were isolated as clumps of cells after exposure to amagnet. These isolated cells were washed once with PBS and transferredto new 60 mm tissue culture dishes containing 5 mL of complete DMEM.These cells were re-screened with 7.5 mg of Dynabeads® magnetic beadscoated with PURE-A for 30 minutes at 4° C. and cells binding to PURE-Awere isolated as clumps of cells after exposure to a magnet (see FIG.5). These re-isolated cell colonies were transferred to 96-well platescontaining 0.25 mL of complete DMEM and the cells were grown in a 37° C.incubator under 5% carbon dioxide until confluent.

To test for the presence of a BoNT/A receptor, individual, cellscontained in the 96-well plates were assayed using the inhibition assayfor insulin release assay, as describes above in Example I, 1a. Celllines containing a candidate BoNT/A receptor were selected based on thedetection of the inhibition of insulin release. FIG. 6 show thattransduced HIT-T15 cell lines C6 and C7 as candidate cell linesexpressing a BoNT/A receptor. To confirm these results, expandedcultures of clones C6 and C7 as described above in Example I, 2a andtested using the inhibition of insulin release assay and the SNAP-25cleavage assay, as described above in Example I, 1 b. The resultsindicate that a BoNT/A receptor is present in these cell lines based onthe inhibition of insulin release (see FIG. 7 a) and the presence of aBoNT/A SNAP25₁₉₇-cleavage product (see FIG. 7 b).

2d. Cloning of BoNT/A Receptor

To isolate nucleic acid molecules encoding the BoNT/A receptor, DNA willbe purified from the BoNT/A receptor-containing HIT-T15 cell isolatesidentified above in Example I, 2c and the nucleic acid molecule encodingthe BoNT/A receptor will be cloned using polymerase chain reaction (PCR)method. Genomic DNA from the C7 cell line will be isolated by analkaline lysis procedure and will be amplified in PCR reactions usingthe ADVANTAGE® Genomic PCR kit (BD Biosciences Clontech, Palo Alto,Calif.) and the following two oligonucleotides5′-AGCCCTCACTCCTTCTCTAG-3′ (SEQ ID NO: 29) and 5′-ACCTACAGGTGGGGTCTTTCATTCCC-3′ (SEQ ID NO: 30). Reactions will be incubated at 95° C. for 1minute, followed by 25 cycles at 68° C. for 30 seconds and 95° C. for 30seconds, followed by 1 cycle at 68° C. for 6 minutes and finalincubation at 4° C. The resulting PCR product will be purified from thePCR reaction by the QIAquick Gel Extraction Kit (QIAGEN, Inc., Valencia,Calif.), and will subjected to a second PCR amplification. Theoligonucleotides used in the second PCR will be nested primers designedto anneal to sequences found within the PCR product originally purified,and will have the following nucleotide sequences:5′-CCCTGGGTCAAGCCCTTTGTACACC-3′ (SEQ ID NO: 31) and 5′-TGCCAAACCTACAGGTGGGGTCTTT-3′ (SEQ ID NO: 32). The resulting nested DNA product willbe subcloned into a pTOPO®-XL vector using the TOPO® TA cloning method(Invitrogen, Inc, Carlsbad, Calif.). The ligation mixture will betransformed into chemically competent E. coli TOP10 cells (Invitrogen,Inc, Carlsbad, Calif.) using a heat shock method, will be plated on 1.5%Luria-Bertani agar plates (pH 7.0) containing 100 μg/mL of Ampicillin,and will be placed in a 37° C. incubator for overnight growth.Ampicillin-resistant colonies will be analyzed using an alkaline lysisplasmid mini-preparation procedure and candidate receptor constructswill be screened by restriction endonuclease mapping to determine thepresence and orientation of the correct insert fragment. Culturescontaining the desired expression construct will be used to inoculate 1L baffled flasks containing 200 mL of Luria-Bertani media containing 100μg/mL of Ampicillin and will be placed in a 37° C. incubator, shaking at250 rpm, for overnight growth. Purified plasmid DNA corresponding to anexpression construct will be isolated using the QIAGEN Maxi-prep method(QIAGEN, Inc., Valencia, Calif.) and will be sequenced to verify thatthe correct expression construct was made (service contract withSequetech Corp., Mountain View, Calif.). This cloning strategy willidentified the sequence composition of the BoNT/A receptor contained inHIT-T15 C7 isolate.

Example II Identification of a BoNT/A Receptor Using a Cross-LinkingProcedure

1. Identification of Cell Lines with High Affinity Uptake for BoNT/A

Distinct sensitivities to each of the BoNT serotypes might be expectedbased on the individual receptor systems for each different toxinserotype and their differing expression in different cell lines. Thepresence of a high affinity receptor system in a cell for BoNT can becharacterized by two attributes: a rapid uptake of the neurotoxin by thecell, and a low neurotoxin concentration needed for cell intoxication.To identify a cell line having a high affinity receptor system for aBoNT/A, we tested cell lines using one of two different in vitrocleavage assay, one to determine the amount of toxin required forintoxication, the other to determine the length of time necessary forthe cell to uptake the neurotoxin.

1a. Assay to Determine the BoNT/A Concentration Necessary for CellIntoxication

In order to assess the amount of BoNT/A needed to intoxicate a cell, apanel of mammalian cell lines of neuronal origin (see Table 3) wasscreened to determine whether toxin exposure would result in thecleavage of endogenously expressed SNAP-25. A suitable seed density ofcells from each line was plated into individual wells of 6-well,poly-D-lysine/Laminin coated, tissue culture plates containing 3 mL of asuitable medium (see Table 3), and grown in a 37° C. incubator under 5%carbon dioxide for approximately 24 hours. BoNT/A (Metabiologics, Inc.,Madison, Wis.) was added at different concentrations (0 nM, 1 nM, 5 nM,12.5 nM, 25 nM, 50 nM) in the culture medium containing the cells forapproximately 8 or approximately 16 hours. Cells were collected in 15 mltubes, washed once with 1 ml of phosphate-buffered saline, pH 7.4, andthen transferred to 1.5 ml microcentrifuge tubes. Cells were lysed in0.5 ml of lysis buffer containing 50 mM N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), pH 6.8, 150 mM sodiumchloride, 1.5 mM magnesium chloride, 1 mM ethylene glycolbis(β-aminoethyl ether) N,N, N′,N′-tetraacetic acid (EGTA), 10% glyceroland 1% (v/v) Triton-X® 100 (4-octylphenol polyethoxylate), with rotationfor 1 hour at 4° C. Lysed cells were centrifuged at 5000 rpm for 10 minat 4° C. to eliminate debris and the supernatants were transferred tofresh siliconized tubes. Protein concentrations were measured byBradford's method and resuspended in 1×SDS sample buffer at 1 mg/ml orhigher concentration.

The presence of a BoNT/A SNAP25₁₉₇-cleavage product was determined byWestern blot analysis as described above in Example I, 1b. A BoNT/ASNAP25₁₉₇-cleavage product was detected in the cell lines SH-SY5Y,NG108-15, N1E-115, Neuro-2A and SK-N-BE(2) after at least an 8 hourincubation with at least 5 nM BoNT/A, thereby indicating the ability ofBoNT/A to intoxicate these cell lines (see FIG. 8 a).

The mouse neuroblastoma cell line Neuro-2A was further analyzed withlower concentrations of BoNT/A to determine the concentration ofneurotoxin necessary to cleave endogenously expressed SNAP-25. Cellswere grown in poly-D-lysine/Laminin coated 6-well plates as describedabove in Example II, 1a. BoNT/A (Metabiologics, Inc., Madison, Wis.) wasadded at different concentrations (0 nM, 0.05 nM, 0.1 nM, 0.2 nM, 0.5nM, 1 nM, 5 nM and 20 nM) in the culture medium containing cells foreither approximately 8 or approximately 16 hours. Toxin treated cellswere harvested and lysed as described above in Example II, 1a. Thepresence of a BoNT/A SNAP25₁₉₇-cleavage product was determined byWestern blot analysis as described above in Example II, 1a. A BoNT/ASNAP25₁₉₇-cleavage product was detected in the cell line Neuro-2A afterat least a 8 hour incubation with at least 0.5 nM BoNT/A, therebyindicating the ability of BoNT/A to intoxicate these cell lines (seeFIG. 8 c).

1b. Assay to Determine the Time Required by a Cell to Uptake BoNT/A

In order to assess the amount of time needed by a cell line to uptakeBoNT/A, a panel of mammalian cell lines of neuronal origin was screenedto determine the length of toxin exposure necessary to cleaveendogenously expressed SNAP-25. Cells from each line were grown inpoly-D-lysine/Laminin coated 6-well plates as described above in ExampleII, 1a. Approximately 1 nM BoNT/A (Metabiologics, Inc., Madison, Wis.)was added to the culture medium for 10 min, 20 min, 30 min, 60 min 2hours, 4 hours, 6 hours, 8 hours or 16 hours. Toxin treated cells werecollected and lysed as described above in Example II, 1a. The presenceof a BoNT/A SNAP25₁₉₇-cleavage product was determined by Western blotanalysis as described above in Example II, 1a. A BoNT/ASNAP25₁₉₇-cleavage product was detected in the cell lines Neuro-2A,SH-SY5Y, and NG108-15 after at least an 8 hour incubation with 1 nMBoNT/A, thereby indicating the ability of these cell lines to rapidlyuptake BoNT/A (see FIG. 8 b).

TABLE 3 Culture Conditions for Cell Lines Cell Line Complete CultureMedia Passage Conditions Seed Density (cells/mm²) SK-N-DZ 90% DMEM, ATrypsin/EDTA treatment, 1:4 dilution split every 2-3 day 4.25 × 10³SK-N-F1 90% DMEM, A Trypsin/EDTA treatment, 1:4 dilution spilt twice aweek 4.25 × 10³ SK-N-SH Ham's F12, DMEM or EMEM, B Trypsin/EDTAtreatment, 1:20 dilution split every 4-7 day 4.25 × 10³ SH-SY5Y EMEM andHam's F12 1:1, C Trypsin/EDTA treatment, 1:6 dilution split every 2-3day 4.25 × 10³ SK-N-BE(2) EMEM and Ham's F12 1:1, D Trypsin/EDTAtreatment, 1:6 dilution split every 3 day 4.25 × 10³ BE(2)-C EMEM andHam's F12 1:1, D Trypsin/EDTA treatment, 1:4 dilution split every 2-3day 4.25 × 10³ BE(2)-M17 EMEM and Ham's F12 1:1, D Trypsin/EDTAtreatment, 1:20 dilution split every 4-7 day 4.25 × 10³ Neuro 2a EMEM, ETrypsin/EDTA treatment, 1:3 dilution split every 3 day 4.25 × 10³ C1300RPMI 1640, B Trypsin/EDTA treatment, 1:3 dilution split every 3 day 4.25× 10³ NB4 1A3 Ham's F10, F Trypsin/EDTA treatment, 1:3 dilution splitevery 3 day 4.25 × 10³ N1E-115 DMEM, G Trypsin/EDTA treatment, 1:3dilution split every 3 day 4.25 × 10³ NG108-15 DMEM, B 1:4 dilutionsplit every 1-2 days 4.25 × 10³ HCN-1A DMEM, H Trypsin/EDTA treatment,1:3 dilution split every 3 day 4.25 × 10³ HCN-2 DMEM, H Trypsin/EDTAtreatment, 1:3 dilution split every 3 day 4.25 × 10³ TE 189.T DMEM, HTrypsin/EDTA treatment, 1:3 dilution split every 3 day 4.25 × 10³ ND8/34DMEM, B Trypsin/EDTA treatment, 1:3 dilution split every 3 day 4.25 ×10³ A contains 1.5 g/L sodium bicarbonate, 0.1 mM Non-essential aminoacids (NEAA), 4 mM Glutamine & 10% Fetal Calf serum (FCS) B contains 2mM Glutamine & 10% FCS C contains 1.5 g/L sodium bicarbonate, 0.1 mMNEAA, 4 mM Glutamine, 1% sodium pyruvate, 1% penicillin/streptomycin(P/S) & 10% FCS D contains 0.1 mM NEAA, 4 mM Glutamine, & 10% FCS Econtains 1.5 g/L sodium bicarbonate, 0.1 mM NEAA, 2 mM Glutamine, 1 mMsodium pyruvate & 10% FCS F contains 2 mM Glutamine, 15% Horse Serum &2.5% FCS G contains 4.5 g/L glucose & 10% FCS H contains 4 mM glucose &10% FCS Freeze medium comprises 95% culture medium and 5% DMSO1c. Ganglioside Treatment to Increase High Affinity Uptake of BoNT/A bya Cell

In order to assess the effect of ganglioside treatment on the ability ofBoNT/A to intoxicate a cell, a Neuro-2A cell line was pre-treated withdifferent gangliosides to determine whether these sugar moieties couldincrease the uptake of BoNT/A by these cells. Neuro-2A cells were platedat a suitable density into individual wells of 6-well,poly-D-lysine/Laminin coated, tissue culture plates containing 3 mL of asuitable medium (see Table 3), and grown in a 37° C. incubator under 5%carbon dioxide. After approximately 24 hours, the medium was replaced bya serum-free media and 25 μg/mL of one of the following gangliosides wasadded to individual wells: GD1a, GD1b, GD3, GQ1b, or GT1b (AXXORA, LLC,San Diego, Calif.). After an overnight 37° C. incubation period, theganglioside-treated cells were washed three times with 1 ml ofphosphate-buffered saline, pH 7.4 and then incubated at 37° C. with 1%serum media containing different concentrations (0 nM, 12.5 nM, 25 nM,50 nM) of BoNT/A (Metabiologics, Inc., Madison, Wis.) for approximately8 or approximately 16 hours. Cells were collected in 15 ml tubes, washedonce with 1 ml of phosphate-buffered saline, pH 7.4, and thentransferred to 1.5 ml microcentrifuge tubes. Cells were lysed in 0.5 mlof lysis buffer containing 50 mM N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), pH 6.8, 150 mM sodiumchloride, 1.5 mM magnesium chloride, 1 mM ethylene glycolbis(β-aminoethyl ether)N,N,N′,N′-tetraacetic acid (EGTA), 10% glyceroland 1% (v/v) Triton-X® 100 (4-octylphenol polyethoxylate), with rotationfor 1 hour at 4° C. Lysed cells were centrifuged at 5000 rpm for 10 minat 4° C. to eliminate debris and the supernatants were transferred tofresh siliconized tubes. Protein concentrations were measured byBradford's method and resuspended in 1×SDS sample buffer at 1 mg/ml orhigher concentration. The presence of a BoNT/A SNAP25₁₉₇-cleavageproduct was determined by Western blot analysis as described above inExample II, 1a. An increase in BoNT/A SNAP25₁₉₇-cleavage product wasdetected in the Neuro-2A cell line treated with the ganglioside GT1b,thereby indicating that GT1b-treatment can increase the uptake of BoNT/Aby Neuro-2A cells (see FIG. 9 a).

1d. Ganglioside Treatment to Increase High Affinity Uptake of BoNT/E bya Cell

In order to assess the effect of ganglioside treatment on the ability ofBoNT/E to intoxicate a cell, a Neuro-2A cell line was pre-treated withdifferent gangliosides to determine whether these sugar moieties couldincrease the uptake of BoNT/E by these cells. Neuro-2A cells were grownin poly-D-lysine/Laminin coated 6-well plates and treated withgangliosides as described above in Example II, 1c. Theganglioside-treated cells were incubated with BoNT/E (Metabiologics,Inc., Madison, Wis.) at different concentrations (0 nM, 12.5 nM, 25 nM,50 nM) in 1% serum media for either approximately 6 or approximately 16hours. Toxin treated cells were harvested and lysed as described abovein Example II, 1c. The presence of a BoNT/E SNAP25₁₈₀-cleavage productwas determined by Western blot analysis as described above in Example I,1b, with the exception that blocked PVDF membranes were incubated in aprimary antibody solution containing a 1:50,000 dilution of mousemonoclonal anti-SNAP-25 antibody (SMI-81; Sternberger Monoclonals,Lutherville, Md.) rather than the rabbit polyclonal anti-SNAP25antiserum pAb anti-SNAP25197 #1 and a secondary antibody solutioncontaining a 1:20,000 dilution of goat polyclonal anti-mouseimmunoglobulin G, heavy and light chains (IgG, H+L) antibody conjugatedto horseradish peroxidase (HRP; Pierce Biotechnology, Inc., Rockford,Ill.) rather than the goat polyclonal anti-rabbit IgG-HRP antibody inorder to detect a BoNT/E SNAP25₁₈₀-cleavage product. An increase inBoNT/E SNAP25₁₈₀-cleavage product was detected in the Neuro-2A celllines treated with the gangliosides GD3, GD1b and GD1a, therebyindicating that GD3-treatment, GD1b-treatment or GD1a-treatment canincrease the uptake of BoNT/E by Neuro-2A cells (see FIG. 9 b).

2. Isolation of BoNT/A Receptor from Neuro-2A Cells

Neuro-2A cells were chosen to conduct ligand cross-linking experimentsusing BoNT/A since these cells had a rapid toxin uptake profile (about10 minutes) and high affinity for BoNT/A. The trifunctional sulfo-SBED(Pierce Biotechnology, Inc., Rockford, Ill.) were used. The reagentsulfo-SBED contains three reactive groups (one of them designed to beUV-activated) and is designed to biotinylate a target protein.

To conjugate a cross-linking agent to a BoNT/A, approximately 100 μg ofPure A is centrifuged at 10,000×g at 4° C. for 10 minutes to pellet thetoxin and brought up in a final volume of 900 μL of phosphate-bufferedsaline (pH 7.4). The solution is then transferred to the dark and 900 μLof 0.25 mM SBED, 1% DMSO solution is added and incubated in a 4° C. fortwo hours in a secondary container on shaking apparatus. The reaction isstopped by adding 50 μL of 1M TRIS (pH 7.4). The solution is inverted 6times and incubated on ice for 30 minutes. The resulting PURE-A-SBEDsolution was used to conduct cross-linking experiments to identify aBoNT/A receptor.

To cross-link PURE-A to BoNT/A receptors present on Neuro-2A cells,about 1.5×10⁵ Neuro-2A cells were plated in a 35 mm tissue culture dishcontaining 3 mL of complete EMEM, supplemented with 10% FBS, 2 mMglutamine (Invitrogen, Inc, Carlsbad, Calif.), 1 mM sodium pyruvate(Invitrogen, Inc, Carlsbad, Calif.), 1.5 g/L sodium bicarbonate and1×MEM non-essential amino acids solution (Invitrogen, Inc, Carlsbad,Calif.), and grown in a 37° C. incubator under 5% carbon dioxide untilthe cells reached a density of about 5×10⁵ cells/ml. The Neuro-2A cellswere harvested by detaching the cells with a trypsin treatment,transferring the cells to 15 ml tubes, and centrifuging the cells at5,000×g at 4° C. for 10 min. The cell pellet is washed three times with9 mL of Tris-buffered saline, and then divided into aliquots of 4×10⁵cells. Each aliquot of cells is suspended in 12 mL cold Tris-bufferedsaline for a final density of 2×10⁷ cells/mL, and placed on ice for 15minutes. To one aliquot of cell suspension, 1 mL of PURE-A-SBED isadded, final concentratin is approximately 100 μg PURE A (33 nM). To asecond cell aliquot, sulfo-SBED only is added and serves as a controlfor false positives. Both Neuro-2 cell suspensions were incubated at 4°C. for two hours in a secondary container using a shaking apparatus andthen each cell solution is distributed in 13 aliquots of 1.0 mL. Thesealiquots were exposed to ultraviolet radiation (365 nm) at 4° C. for 15minutes.

The cells were centrifugation at 5,000×g at 4° C. for 15 minutes andwashed once with 1 mL cold Tris-buffered saline. Washed cells were lysedin 0.5 ml of lysis buffer containing 50 mM N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), pH 6.8, 150 mM sodiumchloride, 1.5 mM magnesium chloride, 1 mM ethylene glycolbis(β-aminoethyl ether)N,N,N′,N′-tetraacetic acid (EGTA), 10% glycerol,1% (v/v) Triton-X® 100 (4-octylphenol polyethoxylate) and suitableprotease inhibitors, with rotation overnight at 4° C. Lysed cells werecentrifuged at 5,000 rpm at 4° C. for 10 min to eliminate debris, thesupernatants were transferred to fresh siliconized tubes and 0.05 mL ofavidin-beads were added to the cleared supernatants. This mixture wasincubated at 4° C. for 3 hours. The avidin beads were then washed twiceby centrifuging at 1000×g at 4° C. for 10 min to pellet beads, decantingthe supernatant, adding 0.5 mL lysis buffer and incubating the solutionat 4° C. for 10 minutes. The avidin beads were then washed twice with0.5 mL phosphate-buffered saline (pH 7.4). Approximately 100 μL ofSDS-PAGE loading buffer was added to the washed, pelleted avidin beadsand boiled for 10 minutes. A 40 μL aliquot was then subjected to MOPSpolyacrylamide gel electrophoresis using NuPAGE® Novex 4-12% Bis-Trisprecast polyacrylamide gels (Invitrogen, Inc, Carlsbad, Calif.) undernon-denaturing and denaturing, reducing conditions. FIG. 10 a shows anapproximately 250 kDa protein in non-reducing gels which represents theintact cross-linking reagent PURE-A-SBED toxin bound to the putativeBoNT/A receptor. Same samples run under denaturing conditions andreveals an approximately 100 kDa protein was co-purified withPURE-A-SBED.

To determine the identity of the BoNT/A receptor isolated from thecross-linking experiments, western blot analysis was performed usingantibodies to the cytoplasmic region of the polypeptides FGF 1 receptor(FGFR1), FGF 2 receptor (FGFR2), FGF 3 receptor (FGFR3) and FGF 4receptor (FGFR4). Approximately 40 μL aliquots of the precipitatedreceptor-PureA complex, obtained as described above in Example II, 2,were separated by MOPS polyacrylamide gel electrophoresis using NuPAGE®Novex 4-12% Bis-Tris precast polyacrylamide gels (Invitrogen, Inc,Carlsbad, Calif.) under non-reducing and denaturing, reducingconditions. Separated peptides were transferred from the gel ontopolyvinylidene fluoride (PVDF) membranes (Invitrogen, Inc, Carlsbad,Calif.) by Western blotting using a Trans-Blot® SD semi-dryelectrophoretic transfer cell apparatus (Bio-Rad Laboratories, Hercules,Calif.). PVDF membranes were blocked by incubating at room temperaturefor 2 hours in a solution containing 25 mM Tris-Buffered Saline (25 mM2-amino-2-hydroxymethyl-1,3-propanediol hydrochloric acid (Tris-HCl) (pH7.4), 137 mM sodium chloride, 2.7 mM potassium chloride), 0.1%TWEEN-20®, polyoxyethylene (20) sorbitan monolaureate, 2% bovine serumalbumin, 5% nonfat dry milk. Blocked membranes were incubated at 4° C.for overnight in Tris-Buffered Saline TWEEN-20® (25 mM Tris-BufferedSaline, 0.1% TWEEN-20®, polyoxyethylene (20) sorbitan monolaureate)containing one of the following primary antibody solutions: 1) a 1:1000dilution of rabbit polyclonal anti-FGFR1 antiserum (Santa CruzBiotechnologies, Inc., Santa Cruz, Calif.); 2) a 1:1000 dilution of goatpolyclonal anti-FGFR2 antiserum (Santa Cruz Biotechnologies, Inc., SantaCruz, Calif.); 3) a 1:1000 dilution of rabbit polyclonal anti-FGFR3(C15) antiserum (Santa Cruz Biotechnologies, Inc., Santa Cruz, Calif.);or 4) a 1:1000 dilution of goat polyclonal anti-FGFR4 antiserum (SantaCruz Biotechnologies, Inc., Santa Cruz, Calif.). Primary antibody probedblots were washed three times for 15 minutes each time in Tris-BufferedSaline TWEEN-20®. Washed membranes were incubated at room temperaturefor 2 hours in Tris-Buffered Saline TWEEN-20® containing either a1:20,000 dilution of goat polyclonal anti-rabbit immunoglobulin G, heavyand light chains (IgG, H+L) antibody conjugated to horseradishperoxidase (HRP; Pierce Biotechnology, Inc., Rockford, Ill.) as asecondary antibody for the FGFR1 and FGFR3 blots or a 1:20,000 dilutionof rabbit polyclonal anti-goat immunoglobulin G, heavy and light chains(IgG, H+L) antibody conjugated to horseradish peroxidase (HRP; PierceBiotechnology, Inc., Rockford, Ill.) for the FGFR2 and FGFR4 blots.Secondary antibody-probed blots were washed three times for 15 minuteseach time in Tris-Buffered Saline TWEEN-20®. Signal detection of thelabeled BoNT/A SNAP25₁₉₇-cleavage product was visualized using the ECLPlus™ Western Blot Detection System (Amersham Biosciences, Piscataway,N.J.) and the membrane was imaged and cleavage product quantitated witha Typhoon 9410 Variable Mode Imager and Imager Analysis software(Amersham Biosciences, Piscataway, N.J.). The choice of pixel size (100to 200 pixels) and PMT voltage settings (350 to 600, normally 400)depended on the individual blot. A band was detected in toxin-receptorsample probed with anti-FGFR3 antiserum of approximately 97 kDa that isconsistent with the size of FGFR3, indicating that FGFR3 is a BoNT/Areceptor (see FIG. 10 b).

3. Identification of BoNT/A Receptor from Various Cells

Several cells lines responsive to BoNT/A uptake were probed withantibodies raised against FGFR1, FGFR2, FGFR3 and FGFR4 in order todetermine which FGFRs these cell lines express. In addition, cells fromthe BoNT/A unresponsive HIT-T15 wild-type cell line and the BoNT/Aresponsive HIT-T15 isolate C7 cell line, as described above in ExampleI, 2c and 2d, were examined. To determine the presence of FGFRs in celllines responsive to BoNT/A exposure, cells were grown, harvested andlysed as described above in Example II, 1a, 1b or 2c and 40 μL aliquotswere subjected to Western blot analysis as described above in ExampleII, 2. These results indicate that the BoNT/A responsive cell linesNeuro-2A, SH-SY5Y and HIT-T15-C7 all express FGFR3, while the BoNT/Aunresponsive wild-type HIT-T15 does not (see FIG. 11). The data alsofrom the revealed that FGFR2 and FGFR4 were not detected in any of thecell lines tested, while FGFR1 was present in all cell lines tested,including wild-type HIT-T15 cells that are unresponsive to BoNT/Aexposure (see FIG. 11).

4. Competitive Competition Assays

To corroborate that BoNT/A toxin enters Neuro-2A cells through the FGFR3we performed a competition experiment with PURE-A and analyzed theresponsiveness of tested using the SNAP-25 cleavage assay, as describedabove in Example I, 1 b. If BoNT/A and an FGFR3 ligand bind to the samereceptor, then increasing amounts of FGF ligand should result indecreased responsiveness of a cell to BoNT/A exposure. However, ifBoNT/A and an FGFR3 ligand bind to the different receptors, thenincreasing amounts of FGF ligand should have no effect of theresponsiveness of a cell to BoNT/A exposure. Table 1, which Applicantsdo not claim is a complete tabulation of FGF receptors and species,shows certain members of the family of FGFRs and their known ligands andtissue distribution.

To determine whether ligands for FGFR3 can competitively compete withBoNT/A for binding to FGFR3, about 5×10⁵ Neuro-2A cells were plated inindividual wells of a 6-well, poly-D-lysine/Laminin coated, tissueculture plates containing 3 mL of EMEM, supplemented with 2 mM glutamine(Invitrogen, Inc, Carlsbad, Calif.), 1 mM sodium pyruvate (Invitrogen,Inc, Carlsbad, Calif.), 1.5 g/L sodium bicarbonate and 1×MEMnon-essential amino acids solution (Invitrogen, Inc, Carlsbad, Calif.),and grown in a 37° C. incubator under 5% carbon dioxide until the cellsreached confluency. Approximately 5 nM PURE-A (Metabiologics, Inc.,Madison, Wis.) was added in conjunction with FGF1, FGF2 or both FGF1 andFGF2 at different concentrations (0 nM, 0.1 nM, 1 nM, 5 nM, 50 nM, 200nM) in the culture medium containing the cells and incubated for at 37°C. for approximately 10 minutes Cells were collected in 15 ml tubes,washed once with 1 ml of phosphate-buffered saline, pH 7.4, and thentransferred to 1.5 ml microcentrifuge tubes. Cells were lysed in 0.5 mlof lysis buffer containing 50 mM N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), pH 6.8, 150 mM sodiumchloride, 1.5 mM magnesium chloride, 1 mM ethylene glycolbis(β-aminoethyl ether)N,N,N′,N′-tetraacetic acid (EGTA), 10% glyceroland 1% (v/v) Triton-X® 100 (4-octylphenol polyethoxylate), with rotationfor 1 hour at 4° C. Lysed cells were centrifuged at 5000 rpm for 10 minat 4° C. to eliminate debris and the supernatants were transferred tofresh siliconized tubes. Protein concentrations were measured byBradford's method and resuspended in 1×SDS sample buffer at 1 mg/ml orhigher concentration.

The presence of a BoNT/A SNAP25₁₉₇-cleavage product was determined byWestern blot analysis as described above in Example II, 1a, with theexception that blocked PVDF membranes will be incubated in a primaryantibody solution containing a 1:50,000 dilution of mouse monoclonalanti-SNAP-25 antibody (SMI-81; Sternberger Monoclonals, Lutherville,Md.) rather than the rabbit polyclonal anti-SNAP-25 antiserum pAbanti-SNAP25197 #1 and a secondary antibody solution containing a1:20,000 dilution of goat polyclonal anti-mouse immunoglobulin G, heavyand light chains (IgG, H+L) antibody conjugated to horseradishperoxidase (HRP; Pierce Biotechnology, Inc., Rockford, Ill.) rather thanthe goat polyclonal anti-rabbit IgG-HRP antibody in order to detect boththe uncleaved SNAP-25 substrate and BoNT/A SNAP25₁₉₇-cleavage product.An increasing amount an increasing amount of FGF ligands, indicatingthese FGF1 and FGF2 compete for the same receptor as BoNT/A and furtherconfirming that FGFR3 is a BoNT/A receptor (see FIG. 12).

Example III

A fusion protein comprising the C terminal portion of the heavy chain ofBoNT/A and the light chain of BoNT/E is tested for its ability toselectively bind and intoxicate BoNT/A susceptible cells. A preparationcomprising dilutions of the fusion protein is incubated with HIT-T15insulinoma cells expressing exogenous FGFR3 in the presence of GT1bganglioside. The ability of the fusion peptide to bind and enter theinsulinoma cells is detected by detecting secretion of insulin inresponse to the presence of glucose, as described above in Example I,1a. By contrast, insulin secretion is unaffected in cells not expressingFGFR3.

The results of this assay show that amount of insulin secreted into theculture medium is decreased in a dose-dependent manner when the fusionprotein is added to the culture medium. Western blots of cell lysateswill show the conversion of full length SNAP-25 to the cleaved formtypical of the proteolytic activity of the BoNT/E light chain protease.This assay therefore is useful in showing that the fusion peptide isable to bind and enter BoNT/A susceptible cells.

The same fusion protein is capable of intoxicating cells of theneuromuscular junction.

Example IV

A fusion protein comprising the receptor binding portion of an FGFspecies capable of binding FGFR3 (including FGF1, FGF2, FGF4 and FGF9)and the translocation domain and light chain of BoNT/E is tested for itsability to selectively bind and intoxicate BoNT/A susceptible cells. Theassay is conducted as described in Example 1 above, with similarresults; the detected cleaved SNAP-25 fragments are characteristic ofBoNT/A intoxication.

Example V

BoNT/A, produced from fermentation of Clostridium botulinum is producedusing standard fermentation techniques. Either or both the bulkpreparation and purified, formulated versions of expressed toxin aretested for purity and activity as follows. A preparation comprisingdilutions of the BoNT/A preparation is incubated with HIT-T15 insulinomacells expressing exogenous FGFR3 in the presence of GT1b ganglioside.The ability of the toxin to bind and enter the insulinoma cells isdetected by detecting secretion of insulin in response to the presenceof glucose, as described above in Example I, 1a. The specific activityof the preparation can be calculated from the determined proteinconcentration and the activity of the preparation at various doses.

These data are submitted to the U.S. Food and Drug Administration by apharmaceutical company as part of data demonstrating how BoNT/A ismanufactured and tested. This information is considered by the FDA, whodecides to permit the manufacture and sale of this lot of BoNT/A, andsubsequent lots made and tested in a similar manner, as a therapeuticpharmaceutical product based in part on this bulk and/or formulationassay data.

The pharmaceutical comprising the BoNT/A is then offered for sale as aprescription medication.

Example VI

Same as Example V, however the polypeptide produced is the fusionneurotoxin of Example III, produced in E. coli. Both bulk and/orformulation lots of the fusion neurotoxin are tested as indicated above,the data submitted to the FDA, and a decision to grant marketingapproval, or continued sales of such fusion polypeptide as a therapeuticagent, is made by the FDA based at least in part on such data. Thepharmaceutical company then offers the fusion neurotoxin for sale as aprescription therapeutic agent.

Example VII

An in vitro assay is established using cloned FGFR3 bound to a solidsupport in the presence of ganglioside GT1b. The bound FGFR3 is firstsaturated with BoNT/A heavy chain (H chain) in phosphate buffered saline(PBS), and washed free of unbound FGF. A test compound from acombinatorial library of compounds is contacted with the receptor undersubstantially physiological conditions (e.g., PBS), and the eluatecollected. The H chain concentration in the eluate is compared to the Hchain concentration of a control eluate in which H chain was not firstbound to FGFR3. Test compounds which are able to strongly bind FGFR3 andcompete with H chain for FGFR3 binding (for example, by the methoddescribed in this section) are candidates compounds for the developmentof an antidote to acute botulism poisoning.

Example VIII Generation of Cells Stably Containing a FGFR3

1. Construction of pQBI25/FGFR3

To construct pQBI-25/FGFR3, a nucleic acid fragment encoding the aminoacid region comprising FGFR3 of SEQ ID NO: 4 is amplified from a humanbrain cDNA library using a polymerase chain reaction method andsubcloned into a pCR2.1 vector using the TOPO® TA cloning method(Invitrogen, Inc, Carlsbad, Calif.). The forward and reverseoligonucleotide primers used for this reaction are designed to includeunique restriction enzyme sites useful for subsequent subcloning steps.The resulting pCR2.1/FGFR3 construct is digested with restrictionenzymes that 1) excise the insert containing the entire open readingframe encoding the FGFR3; and 2) enable this insert to beoperably-linked to a pQBI-25 vector (Qbiogene, Inc., Irvine, Calif.).This insert is subcloned using a T4 DNA ligase procedure into a pQBI-25vector that is digested with appropriate restriction endonucleases toyield pQBI-25/FGFR3. The ligation mixture is transformed into chemicallycompetent E. coli BL21 (DE3) cells (Invitrogen, Inc, Carlsbad, Calif.)using a heat shock method, plated on 1.5% Luria-Bertani agar plates (pH7.0) containing 100 μg/mL of Ampicillin, and placed in a 37° C.incubator for overnight growth. Bacteria containing expressionconstructs are identified as Ampicillin resistant colonies. Candidateconstructs are isolated using an alkaline lysis plasmid mini-preparationprocedure and analyzed by restriction endonuclease digest mapping todetermine the presence and orientation of the inset. This cloningstrategy yields a mammalian expression construct encoding the FGFR3 ofSEQ ID NO: 4 operably-linked to the expression elements of the pQBI-25vector.

2. Stably Transformed Cells Using a Recombinant Crossing-Over Procedure

To generate a stably-integrated cell line expressing a FGFR3 using acrossing over procedure, a suitable density (1×10⁵ to 1×106⁶ cells) ofappropriate cells, such as, e.g., HIT-T15 or Neuro2A, are plated in a 35mm tissue culture dish containing 3 mL of complete, supplemented culturemedia and grown in a 37° C. incubator under 5% carbon dioxide until thecells reached a density appropriate for transfection. A 500 μLtransfection solution is prepared by adding 250 μL of OPTI-MEM ReducedSerum Medium containing 15 μL of LipofectAmine 2000 (Invitrogen,Carlsbad, Calif.) incubated at room temperature for 5 minutes to 250 μLof OPTI-MEM Reduced Serum Medium containing 5 μg of expression constructencoding a FGFR3, such as, e.g., pQBI-25/FGFR3 (see Examples VIII, 1).This transfection was incubated at room temperature for approximately 20minutes. The complete, supplemented media is replaced with 2 mL ofOPTI-MEM Reduced Serum Medium and the 500 μL transfection solution isadded to the cells and the cells are incubated in a 37° C. incubatorunder 5% carbon dioxide for approximately 16 hours. Transfection mediais replaced with 3 mL of fresh complete, supplemented culture media andthe cells are incubated in a 37° C. incubator under 5% carbon dioxidefor approximately 48 hours. Media is replaced with 3 mL of freshcomplete, supplemented culture media, containing approximately 5 μg/mLof G418. Cells are incubated in a 37° C. incubator under 5% carbondioxide for approximately 4 weeks, with old media being replaced withfresh G418 selective, complete, supplemented media every 4 to 5 days.Once G418-resistant colonies are established, resistant clones arereplated to new 35 mm culture plates containing fresh complete culturemedia, supplemented with approximately 5 μg/mL of G418 until these cellsreached a density of 6 to 20×10⁵ cells/mL.

To test for expression of a FGFR3 from isolated cell lines thatstably-integrated an expression construct encoding a FGFR3, such as,e.g., pQBI-25/FGFR3 (see Examples VIII, 1), approximately 1.5×10⁵ cellsfrom each cell line are plated in a 35 mm tissue culture dish containing3 mL of G418-selective, complete, supplemented DMEM and are grown in a37° C. incubator under 5% carbon dioxide until cells reached a densityof about 5×10⁵ cells/ml (6-16 hours). Media is replaced with 3 mL offresh G418-selective, complete, supplemented culture media and cells areincubated in a 37° C. incubator under 5% carbon dioxide. After 48 hours,the cells are harvested by rinsing the cells once with 3.0 mL of 100 mMphosphate-buffered saline, pH 7.4 and are lysed with a buffer containing62.6 mM 2-amino-2-hydroxymethyl-1,3-propanediol hydrochloric acid(Tris-HCl), pH 6.8 and 2% sodium lauryl sulfate (SDS). Lysed cells arecentrifuged at 5000 rpm for 10 min at 4° C. to eliminate debris and thesupernatants are transferred to fresh siliconized tubes. Proteinconcentrations are measured by Bradford's method and are resuspended in1×SDS sample buffer at 1 mg/ml or higher concentration.

To detect for the presence of a FGFR3, samples are separated by MOPSpolyacrylamide gel electrophoresis and analyzed by Western blottingprocedures as described above in Example II, 2 using a 1:1000 dilutionof rabbit polyclonal anti-FGFR3 (C15) antiserum (Santa CruzBiotechnologies, Inc., Santa Cruz, Calif.), in order to identify celllines that have stably integrated and express the FGFR3 substrate.

Example IX FGFR3 Phosphorylation Studies

1. Phosphorylation of FGFR-3 Exposed to FGF or BoNT/A

When bound by specific ligands, FGFR's are auto-phosphorylated onspecific tyrosine residues. This begins the process of internalizationof both the receptor and the ligand into the endosomal pathway. IfBoNT/A binds to FGFR3, then exposure to BoNT/A should cause theauto-phosphorylation of FGFR3 in exposed cells.

To determine whether BoNT/A binding resulted in FGFR3 phosphorylation,approximately 1.5×10⁵ Neuro-2A cells were plated into the wells of6-well, poly-D-lysine/Laminin coated, tissue culture plates containing 3mL of serum-free EMEM, supplemented with 1 mM sodium pyruvate(Invitrogen, Inc, Carlsbad, Calif.), 1.5 g/L sodium bicarbonate and1×MEM non-essential amino acids solution (Invitrogen, Inc, Carlsbad,Calif.), and grown in a 37° C. incubator under 5% carbon dioxide untilthe cells reached a density of about 5×10⁵ cells/ml. The serum-freemedia was replaced with fresh supplemented EMEM containing 1% FBS(Invitrogen, Inc, Carlsbad, Calif.) and either 5 nM FGF-2 (BiosourceInternational, Camarillo, Calif.) or 5 nM of PURE/A (Metabiologics,Inc., Madison, Wis.). The cells were then incubated in a 37° C.incubator under 5% carbon dioxide for approximately 5 min, 10 min, 20min and 30 min, with unexposed cells used as time 0. Cells werecollected in 15 ml tubes, washed once with 1 ml of phosphate-bufferedsaline, pH 7.4, and then transferred to 1.5 ml microcentrifuge tubes.Cells were lysed in 0.5 ml of lysis buffer containing 50 mMN-(2-hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid) (HEPES), pH6.8, 150 mM sodium chloride, 1.5 mM magnesium chloride, 1 mM ethyleneglycol bis(β-aminoethyl ether) N,N, N′,N′-tetraacetic acid (EGTA), 10%glycerol and 1% (v/v) Triton-X® 100 (4-octylphenol polyethoxylate), withrotation for 1 hour at 4° C. Lysed cells were centrifuged at 5000 rpmfor 10 min at 4° C. to eliminate debris and the supernatants weretransferred to fresh siliconized tubes. Protein concentrations weremeasured by Bradford's method and resuspended in 1×SDS sample buffer at1 mg/ml or higher concentration.

Supernatant containing 100 μg of protein was immunoprecipitated using 5μg of anti-phosphotyrosine antibody attached to a sepharose bead (ZymedLaboratories, Inc., South San Francisco, Calif.). The immunoprecipitatedproduct were subjected to Western blot analysis as described above inExample II, 4, with the blots being probed for FGFR3 (Santa CruzBiotechnologies, Inc., Santa Cruz, Calif.). These experiments show thatFGFR3 is phosphorylated upon either FGF2 or BoNT/A exposure, indicatingthat BoNT/A binds to FGFR3 (see FIG. 13 a).

2. DMBI Inhibition of FGFR-3 Phosphorylation Exposed to FGF

To determine whether DMBI inhibits BoNT/A-induced FGFR3 phosphorylation,Neuro-2A cells were plated and grown as described above in ExampleIX, 1. Neuro-2A cells were plated at a density of 5×10⁵ cells/well (6well plate) and incubated overnight in serum-free media. The media wasreplaced with fresh serum-free supplemented EMEM containing 0, 1 μM, 5μM, 20 μM, or 100 μM of DMBI (EMD Calbiochem, San Diego, Calif.) for 1hour. DMBI inhibits the autophosphorylation and dimerization of FGFR andPDGF type receptors. The cells were then washed and fresh supplementedEMEM containing 1% FBS (Invitrogen, Inc, Carlsbad, Calif.) and 5 nMFGF-2 (Biosource International, Camarillo, Calif.). The cells were thenincubated in a 37° C. incubator under 5% carbon dioxide forapproximately 5 min, 10 min and harvested and immunoprecipitated asdescribed above in Example IX, 1. The immunoprecipitated products weresubjected to Western blot analysis as described above in Example II, 4,with the exception that the blots were probed with a primary antibodysolution containing a 1:1000 dilution of a rabbit polyclonalanti-phosphotyrosine antiserum (Upstate USA, Inc., Charlottesville, Va.)and a secondary antibody solution containing a 1:20,000 dilution of goatpolyclonal anti-rabbit immunoglobulin G, heavy and light chains (IgG,H+L) antibody conjugated to horseradish peroxidase (HRP; PierceBiotechnology, Inc., Rockford, Ill.). These results indicate that DMBIeffectively inhibits the phosphorylation of FGFR3 upon FGF2 exposure(see FIG. 13 b).

3. DMBI Inhibition of BoNT/A Activity

To determine whether DMBI can inhibit BoNT/A activity, Neuro-2A cellswere plated and grown as described above in Example IX, 1. The media wasreplaced with fresh serum-free supplemented EMEM containing 0, 1 μM, 5μM, 20 μM, or 100 μM of DMBI (EMD Calbiochem, San Diego, Calif.) for 1hour. DMBI inhibits the autophosphorylation and dimerization of FGFR andPDGF type receptors. The cells were then washed and fresh supplementedEMEM containing 1% FBS (Invitrogen, Inc, Carlsbad, Calif.) and 5 nM ofPURE/A (Metabiologics, Inc., Madison, Wis.). The cells were thenincubated in a 37° C. incubator under 5% carbon dioxide forapproximately 5 min, 10 min and harvested as described above in ExampleIX, 1. Aliquots were tested for the presence of the BoNT/A SNAP-25₁₉₇cleavage product using the SNAP-25 cleavage assay as described above inExample I, 1 b. These results indicate a reduction in the amount ofSNAP-25 cleavage product present, thereby indicating that DMBIeffectively inhibits BoNT/A activity and confirming that this toxin ininternalized by FGFR3 (see FIG. 13 c).

The examples provided herein are simply illustrations of various aspectsof the invention, which is to be understood to be defined solely by theclaims which follow this specification.

1. An in vitro method of screening for a molecule able to compete with botulinum neurotoxin serotype A (BoNT/A) for selective binding to cells susceptible to BoNT/A intoxication, the method comprising the steps of: a) contacting a sample containing a molecule and a BoNT/A with a cell composition comprising an exogenous Fibroblast Growth Factor Receptor 3 (FGFR3); and b) detecting whether said molecule is able to compete with BoNT/A for the selective binding to said FGFR3 wherein selective binding of said molecule to said FGFR3 indicates that said molecule is able to compete with BoNT/A for selective binding to cells susceptible to BoNT/A intoxication, and wherein if said molecule is BoNT/A, said method does not comprise an LD50 assay, wherein said composition is a cell composition, and wherein said cell composition contains an exogenous FGFR3, wherein said cell composition is genetically engineered to express a nucleic acid molecule encoding said FGFR3.
 2. The method according to claim 1, wherein said cell composition comprises a non-neuronal cell, said non-neuronal cell expressing an endogenous Synaptosomal-Associated Protein 25 kDA (SNAP-25).
 3. The method according to claim 2, wherein said non-neuronal cell is a primary non-neuronal cell or an immortalized non-neuronal cell.
 4. The method according to claim 2, wherein said non-neuronal cell is selected from the group consisting of an anterior pituitary cell, an adrenal cell, a pancreatic cell, an ovarian cell, a kidney cell, a stomach cell, a blood cell, an epithelial cell, a fibroblast, a thyroid cell, a chondrocyte, a muscle cell, a hepatocyte, a glandular cell. 